Process for formulating an anionic agent

ABSTRACT

Formulations comprising anionic agents such as nucleic acids within a lipid-containing particle methods of formulating a lipid-containing particle comprising an anionic agent such as a nucleic acid, methods for preparing a lipid-containing particle comprising an anionic agent such as a nucleic acid, methods for therapeutic delivery of an anionic agent to a patient in need thereof, where the anionic agent is formulated in a lipid-containing particle as described herein.

CROSS REFERENCE

This application is a continuation of PCT International ApplicationSerial Number PCT/US14/29372, filed Mar. 14, 2014, designating theUnited States, claiming priority from U.S. Provisional Application No.61/784,810 entitled “Process for Formulating an Anionic Agent” and filedMar. 14, 2013, incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

Nucleic acid molecules cannot easily cross cell membranes because oftheir size and hydrophilicity. Delivery has therefore been one of themajor challenges for nucleic acid therapeutics, e.g., antisense payloadsand RNAi technology. To trigger RNase H activity or RNAi activityfollowing systemic administration, a formulation containing nucleic acidmolecules not only must (1) protect the payload from enzymatic andnon-enzymatic degradation and (2) provide appropriate biodistribution ofthe formulation, but also (3) allow cellular uptake or internalizationof the formulation and (4) facilitate delivery of the nucleic acidpayload to the cytoplasm of the cell. Many formulations that excel incriteria 1 and 2 above are deficient in criteria 3 and 4, and manynucleic acid formulations therefore show excellent biodistribution butfail to knock down the target gene due to lack of systemic delivery andlocal delivery.

While a number of lipid-based formulations have recently beendemonstrated to effect intracellular delivery of nucleic acid payloadsto at least certain types of mammalian cells (e.g., mammalian livercells), the precise proportions and methods of combining lipids,payloads and other components of such formulations can greatly influencethe extent to which successful delivery of nucleic acid payloads isachieved. Accordingly, modest changes in the processes employed toobtain such lipid-based formulations have the potential to producedramatic and surprising differences in delivery efficacy. As such, thereis a need to optimize the process by which lipid-based formulations ofnucleic acid payloads (and, by extension, anionic agents more generally)are obtained, thereby enhancing the delivery of such therapeutic anionicagents to cells.

SUMMARY OF THE INVENTION

The invention relates, at least in part, to methods of formulatinganionic agents, e.g., anionic therapeutic agents such as nucleic acids.In particular aspects of the invention, a process for preparing alipid-containing particle comprising an anionic agent (e.g., a nucleicacid payload) has been identified, which involves combining a lipidcomplex with the anionic agent under conditions in which, due to theorder of addition of such components, the total lipid solventconcentration of the mixed solution increases or is held stable overtime, rather than declines, resulting in a formulated particle thatpossesses improved structural homogeneity and improved efficacy ofintracellular delivery of the anionic agent. (While not wishing to bebound by theory, improved structural homogeneity appears to result froma reduction in lipidic complex dissolution during the mixing process, ascompared to processes that produce a decline in lipid solventconcentration during the process of mixing the lipid complex and theanionic agent.) Without wishing to be bound by theory, at least oneadvantage of the methods of the instant invention is that they are morescalable than other processes, allowing for improved particleformation/formulation in amounts sufficient for, e.g., performance ofclinical trials and/or commercial sale.

In other aspects of the invention, processes are provided which arebased upon the surprising observation that a relatively low solubilitylimit possessed by an individual lipid or sterol in a solvent (e.g.,ethanol or alcohol or organic solution or mixture thereof) can beeffectively raised at room temperature by mixing other lipid(s) and/orsterol(s) together in a solvent (or, optionally, simply in a neat lipidoil or mixture of lipid oils) before adding this mixture of otherlipid(s) and/or sterol(s) in oil or solvent to the relatively lowsolubility lipid or sterol, optionally further mixing such lipid and/orsterol suspension into the solvent. For example, while the solubilitylimit of cholesterol in ethanol at room temperature was observed to beabout 10-11 mg/ml in the absence of other lipids and/or sterols, it wasunexpectedly discovered that a pre-mixing of additional lipids asdescribed herein in ethanol before addition of such a lipid suspensionin ethanol to cholesterol (as a powder) at room temperature allowed forcholesterol levels of 20 mg/ml or higher to be achieved in the solution,while the total lipid content of such solutions could also be raised to37 mg/ml, 74 mg/ml, or even higher levels. Thus, a process is providedfor raising the amount of an original lipid, sterol and/or blend oflipid(s) and/or sterol(s) that may be solubilized in a solvent (e.g., analcoholic solvent, e.g., ethanol) by pre-mixing other lipid(s),sterol(s) and/or blend of lipid(s) and/or sterol(s), as oils, powdersand/or in solvent(s), before adding such a pre-mixture to the originalsolubility-limited lipid, sterol and/or blend of lipid(s), therebyeffectively raising the solubility limit of the original lipid in thesolvent. In certain related embodiments, the invention provides aprocess for making a particle that involves pre-mixing elevatedconcentrations of lipid and/or sterol components as oils, powders and/orin solvent (e.g., ethanol), adding this mixture to a lipid and/or sterolthat possesses relatively low solubility in the solvent in the absenceof such pre-mixed lipid(s) and/or sterol(s), and combining this mixturewith anionic agent-containing aqueous solutions or suspensions(optionally, such anionic agents are complexed with lipid prior to suchaddition of solvent-suspended lipids). Without wishing to be bound bytheory, it is believed that the newly discovered ability to provide highconcentration solutions of lipids/sterols at such elevatedconcentrations enhances the homogeneity of a lipid-anionic agentparticle population, as compared to the concentrations at which suchlipids/sterols are routinely used within the particle formulationprocess.

In one aspect, the invention provides a method of producing a particleharboring an anionic agent that involves combining a modified lipidwhich prevents particle aggregation during lipid-anionic agent particleformation with a cationic lipid in an acidic aqueous solution, in anamount sufficient for a complex to form; combining this complex with ananionic agent; combining the complex-anionic agent with a neutralaqueous solution to form a complex-anionic agent aqueous suspension;forming a solution or suspension that includes at least one structurallipid, sterol, cationic lipid or modified lipid which prevents particleaggregation during lipid-anionic agent particle formation; and combiningthis solution or suspension with the complex-anionic agent aqueoussolution of the previous step by a method that either involves addingthe solution or suspension to the complex-anionic agent aqueoussuspension or in-line mixing the solution or suspension and thecomplex-anionic agent aqueous solution.

In certain embodiments, the acidic aqueous solution includes HCl.Optionally, the acidic aqueous solution possesses a pH of less than 4,in certain embodiments, 2.3. In related embodiments, the acidic aqueoussolution is about 60 mM HCl.

In one embodiment, the cationic lipid that is present in acidic aqueoussolution possesses a protonatable group. Optionally, this cationic lipidhas a pKa of 4 to 11. In certain embodiments, this cationic lipid isDODMA, DOTMA, or a cationic lipid of Table 1.

In certain embodiments, the modified lipid which prevents particleaggregation during lipid-anionic agent particle formation is aPEG-lipid, optionally DMPE-PEG, DSPE-PEG or DSG-PEG. In relatedembodiments, the PEG is PEG2k.

In some embodiments, the modified lipid-cationic lipid complex isbetween 60 and 75 nM in diameter.

In certain embodiments, the anionic agent is a polyanionic agent. Inrelated embodiments, the anionic agent is a nucleic acid. Optionally,the nucleic acid is an antisense oligonucleotide or a double-strandednucleic acid. In certain embodiments, the double-stranded nucleic acidis a small hairpin RNA (shRNA) or a siRNA. In a related embodiment, thedouble-stranded nucleic acid is a substrate for human Dicer and isoptionally a DsiRNA.

In some embodiments, the neutral aqueous solution is water.

In certain embodiments, forming a solution or suspension that includesat least one structural lipid, sterol, cationic lipid or modified lipidwhich prevents particle aggregation during lipid-anionic agent particleformation involves dissolving in ethanol the at least one lipid.Optionally, this forming of a solution or suspension involves dissolvingthe lipid or sterol in 100% ethanol. In related embodiments thestructural lipid is DSPC, DPPC or DOPC. In certain embodiments, thesterol is cholesterol. Optionally, the cationic lipid is selected fromTable 1.

In certain embodiments, the particle harboring an anionic agent isbetween 90 and 110 nm in diameter.

In one embodiment, the particle harboring an anionic agent is made at ascale of 10 mg or more of anionic agent, 50 mg or more of anionic agent,100 mg or more of anionic agent, 250 mg or more of anionic agent, 500 mgor more of anionic agent, 1 g or more of anionic agent, 2 g or more ofanionic agent, 3 g or more of anionic agent, 4 g or more of anionicagent, 5 g or more of anionic agent, 7.5 g or more of anionic agent, 10g or more of anionic agent, 20 g or more of anionic agent, 40 g or moreof anionic agent, 50 g or more of anionic agent, 100 g or more ofanionic agent, 200 g or more or anionic agent, 300 g or more of anionicagent, 400 g or more of anionic agent, 500 g or more of anionic agent, 1kg or more of anionic agent, 2 kg or more of anionic agent, 3 kg or moreof anionic agent, 4 kg or more of anionic agent, 5 kg or more of anionicagent or 10 kg or more of anionic agent.

In another embodiment, the particle harboring an anionic agent possessesone or more of the following properties: improved size and/or PDI,improved efficacy in a subject administered the particle or improvedtolerability in a subject administered the particle, as compared to anappropriate control particle formed by an appropriate control processthat involves adding the complex-anionic agent aqueous suspension to thesolvent-based solution or suspension.

In an additional embodiment, the method further involves combining theparticle harboring an anionic agent with a volume of water sufficient toreduce the concentration of ethanol within the combined solution to 10%or less.

In another embodiment, the method also involves performing one of thefollowing processes: tangential flow filtration (TFF) or dialysis uponthe combined solution. Optionally, the combined solution is dialyzedagainst PBS.

Another aspect of the invention provides a method of producing aparticle harboring an anionic agent which involves combining in anacidic aqueous solution a modified lipid which prevents particleaggregation during lipid-anionic agent particle formation with acationic lipid, in an amount sufficient for a complex to form; combiningthis complex with an anionic agent; combining this complex-anionic agentwith a neutral aqueous solution to form a complex-anionic agent aqueoussuspension; forming a solution or suspension having at least one of astructural lipid, a sterol, a cationic lipid or a modified lipid whichprevents particle aggregation during lipid-anionic agent particleformation; and adding this solution or suspension to the complex-anionicagent aqueous suspension.

An additional aspect of the invention provides a method for increasingthe solubility of a first lipid or sterol in a solvent which involvescombining a second lipid or sterol with the solvent to form a secondlipid or sterol solution in the solvent, where the solvent is free ofthe first lipid; and combining the second lipid or sterol solution inthe solvent with the first lipid or sterol to form a solution of thesecond lipid or sterol and the first lipid or sterol, where thesolubility of the first lipid or sterol in the solvent in the presenceof the second lipid or sterol is higher than the solubility of the firstlipid or sterol in the solvent in the absence of the second lipid orsterol.

A further aspect of the invention provides a method for increasing thesolubility of a first lipid or sterol in a solvent which involvescombining a second lipid or sterol with the first lipid or sterol in theabsence of a solvent, where the solubility of the first lipid or sterolin the solvent in the presence of the second lipid or sterol is higherthan the solubility of the first lipid or sterol in the solvent in theabsence of the second lipid or sterol.

In one embodiment, the first lipid or sterol is a sterol, optionallycholesterol, cholestanone, cholestenone, coprostanol,3β-[-(N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol(DC-cholesterol) or bis-guanidium-tren-cholesterol (BGTC).

In another embodiment, the first lipid or sterol is present at aconcentration of 10 mg/ml or more, 11 mg/ml or more, 12 mg/ml or more,15 mg/ml or more, 20 mg/ml or more, 25 mg/ml or more, 30 mg/ml or more,35 mg/ml or more, 37 mg/ml or more, 40 mg/ml or more, 45 mg/ml or more,50 mg/ml or more, 55 mg/ml or more, 60 mg/ml or more, 65 mg/ml or more,70 mg/ml or more, 74 mg/ml or more, 75 mg/ml or more, 80 mg/ml or more,85 mg/ml or more, 90 mg/ml or more, 95 mg/ml or more, 100 mg/ml or more,150 mg/ml or more, 200 mg/ml or more, 250 mg/ml or more, 500 mg/ml ormore or 1 g/ml or more within the solution of the second lipid or steroland the first lipid or sterol.

In one embodiment, the total lipid content of the solution of the secondlipid or sterol and the first lipid or sterol is 12 mg/ml or more, 15mg/ml or more, 20 mg/ml or more, 25 mg/ml or more, 30 mg/ml or more, 35mg/ml or more, 37 mg/ml or more, 40 mg/ml or more, 45 mg/ml or more, 50mg/ml or more, 55 mg/ml or more, 60 mg/ml or more, 65 mg/ml or more, 70mg/ml or more, 74 mg/ml or more, 75 mg/ml or more, 80 mg/ml or more, 85mg/ml or more, 90 mg/ml or more, 95 mg/ml or more, 100 mg/ml or more,150 mg/ml or more, 200 mg/ml or more, 250 mg/ml or more, 500 mg/ml ormore or 1 g/ml or more.

In one embodiment, the second lipid or sterol solution in the solventincludes one or more additional lipids of Tables 1-4.

In another embodiment, the solution of the second lipid or sterol andthe first lipid or sterol includes at least one of a structural lipid, asterol, a cationic lipid or a modified lipid which prevents particleaggregation during lipid-anionic agent particle formation.

Another aspect of the invention provides a method of producing aparticle having a first lipid or sterol, a second lipid or sterol and asmall molecule, which involves combining a second lipid or sterol with asolvent to form a second lipid or sterol solution in the solvent, wherethe solvent is free of the first lipid or sterol; and combining thesecond lipid or sterol solution in the solvent with the first lipid orsterol to form a solution of the second lipid or sterol and the firstlipid or sterol, where the solubility of the first lipid or sterol inthe solvent in the presence of the second lipid or sterol is higher thanthe solubility of the first lipid or sterol in the solvent in theabsence of the second lipid or sterol, then combining this solution ofthe second lipid or sterol and the first lipid or sterol with a smallmolecule.

An additional aspect of the invention provides a method of producing aparticle having a first lipid or sterol, a second lipid or sterol and ananionic agent, which involves combining a second lipid or sterol with asolvent to form a second lipid or sterol solution in the solvent, wherethe solvent is free of the first lipid or sterol; and combining thesecond lipid or sterol solution in the solvent with the first lipid orsterol to form a solution of the second lipid or sterol and the firstlipid or sterol, where the solubility of the first lipid or sterol inthe solvent in the presence of the second lipid or sterol is higher thanthe solubility of the first lipid or sterol in the solvent in theabsence of the second lipid or sterol, then combining this solution ofthe second lipid or sterol and the first lipid or sterol with an anionicagent.

In a further aspect, the invention provides a method of producing aparticle having a first lipid or sterol, a second lipid or sterol and ananionic agent, which involves combining in an acidic aqueous solution amodified lipid which prevents particle aggregation during lipid-anionicagent particle formation and a cationic lipid, in an amount sufficientfor a complex to form; combining this complex with an anionic agent;combining a neutral aqueous solution with the complex-anionic agent toform a complex-anionic agent aqueous suspension; combining a secondlipid or sterol with a solvent to form a second lipid or sterol solutionin the solvent, where the solvent is free of the first lipid or sterol;combining the second lipid or sterol solution in the solvent with thefirst lipid or sterol to form a solution of the second lipid or steroland the first lipid or sterol; and combining the solution of the secondlipid or sterol and the first lipid or sterol with the complex-anionicagent aqueous solution.

In certain embodiments, the solubility of the first lipid or sterol inthe solvent in the presence of the second lipid or sterol is higher thanthe solubility of the first lipid or sterol in the solvent in theabsence of the second lipid or sterol.

In one embodiment, the solution of the second lipid or sterol and thefirst lipid or sterol includes at least one of a structural lipid, asterol, a cationic lipid or a modified lipid which prevents particleaggregation during lipid-anionic agent particle formation.

In another embodiment, the particle possesses at least one of thefollowing properties: improved size and/or PDI, improved efficacy in asubject administered the particle or improved tolerability and/orreduced toxicity in a subject administered the particle, as compared toan appropriate control particle formed by an appropriate control processthat involves exposing the first lipid or sterol to the solvent beforethe second lipid or sterol is exposed to the solvent.

In certain embodiments, the solution of the second lipid or sterol andthe first lipid or sterol is added to the complex-anionic agent aqueoussuspension or the solution of the second lipid or sterol and the firstlipid or sterol is in-line mixed with the complex-anionic agent aqueoussolution.

In certain embodiments, it is also contemplated that the solubility of afirst lipid or sterol in a solvent can be effectively raised byinitially adding the first lipid or sterol to the solvent at aconcentration below the solubility limit of the first lipid or sterol,then adding a second lipid or sterol to the solution, and then addingthe first lipid or sterol to the solution, such that the first lipid orsterol is added to achieve a concentration in the secondlipid/sterol-containing solution that exceeds the original solubilitylimit of the first lipid or sterol in the solvent absent such additionof the second lipid or sterol.

In one aspect, the invention features a compound (e.g., lipid orcationic lipid) having the formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl, where theR¹ and R² is not substituted with an oxo on the carbon adjacent to>CHNR³R⁴; R³ is H or optionally substituted C₁₋₆ alkyl; and R⁴ isunsubstituted C₁₋₆ alkyl that is substituted with —NR^(4a)R^(4b),substituted C₁₋₆ alkyl that is further substituted with —NR^(4a)R^(4b),or optionally substituted C₃₋₇ heterocyclyl, where each R^(4a) andR^(4b) is, independently, H, C(═NH)NH₂, or optionally substituted C₁₋₆alkyl, or where R^(4a) and R^(4b) combine together to form optionallysubstituted C₃₋₇ heterocyclyl; and where R³ and R⁴ can combine togetherto form an optionally substituted C₃₋₇ heterocyclyl; where R³ and R⁴ donot combine together to form optionally substituted imidazolyl oroptionally substituted benzimidazolyl or optionally substitutedsuccinimidyl; where one, and only one, primary amine can be present oneither R³ or R⁴ or no primary amine is present on either R³ or R⁴; andwhere neither R³ nor R⁴ is an optionally substituted amide; and wherewhen R¹ or R² is saturated C₁₁ alkyl or saturated C₁₅ alkyl, R³ is notH; where when R¹ or R² is saturated C₁₆ alkyl or saturated C₁₇ alkyl, R¹and R² is not substituted with hydroxy; where when R¹ or R² is saturatedC₁₇ alkyl, R³ or R⁴ is not substituted with hydroxy; and where when R¹or R² is saturated C₁₈ alkyl, R⁴ is not substituted with optionallysubstituted imidazolyl.

In some embodiments, R³ is C₁₋₆ alkyl substituted with —NR^(3a)R^(3b)and where each R^(3a) and R^(3b) is, independently, H or optionallysubstituted C₁₋₆ alkyl. In particular embodiments, each R^(3a) andR^(3b) is, independently, H or C₁₋₆ alkyl.

In some embodiments, R⁴ is unsubstituted C₁₋₆ alkyl that is substitutedwith —NR^(4a)R_(4b). In particular embodiments, R⁴ is substituted C₁₋₆alkyl (e.g., substituted C₁₋₃ alkyl, substituted C₁₋₂ alkyl, substitutedC₁ alkyl, substituted C₂ alkyl, or substituted C₃ alkyl) or C₁₋₆aminoalkyl that is further substituted with —NR^(4a)R^(4b). In someembodiments, R⁴ is C₁₋₆ alkyl substituted with an oxo and is furthersubstituted with —NR^(4a)R^(4b). In some embodiments, R^(4a) and R^(4b)combine together to form an optionally substituted C₃₋₇ heterocyclyl(e.g., optionally substituted pyrrolidinyl, optionally substitutedimidazolidinyl, optionally substituted pyrazolidinyl, optionallysubstituted piperidinyl, optionally substituted piperazinyl, optionallysubstituted azepanyl, optionally substituted pyrrolyl, optionallysubstituted imidazolyl, or optionally substituted pyrazolyl). In someembodiments, each R^(4a) and R^(4b) is, independently, optionallysubstituted C₁₋₆ alkyl. In some embodiments, R⁴ is unsubstituted C₁₋₆alkyl that is substituted with optionally substituted C₃₋₇ heterocyclyl(e.g., any described herein). In some embodiments, R⁴ is substitutedC₁₋₆ alkyl (e.g., with an oxo) or a C₁₋₆ aminoalkyl that is furthersubstituted with optionally substituted C₃₋₇ heterocyclyl (e.g.,optionally substituted pyrrolidinyl, optionally substitutedimidazolidinyl, optionally substituted pyrazolidinyl, optionallysubstituted piperidinyl, optionally substituted piperazinyl, optionallysubstituted azepanyl, optionally substituted pyrrolyl, optionallysubstituted imidazolyl, optionally substituted pyrazolyl, optionallysubstituted pyridinyl, optionally substituted pyrazinyl, optionallysubstituted pyrimidinyl, or optionally substituted pyridazinyl).

In some embodiments, R³ and R⁴ combine together to form an optionallysubstituted C₃₋₇ heterocyclyl (e.g., optionally substitutedpyrrolidinyl, optionally substituted imidazolidinyl, optionallysubstituted pyrazolidinyl, optionally substituted piperidinyl,optionally substituted piperazinyl, optionally substituted azepanyl,optionally substituted pyrrolyl, optionally substituted imidazolyl,optionally substituted pyrazolyl, optionally substituted pyridinyl,optionally substituted pyrazinyl, optionally substituted pyrimidinyl, oroptionally substituted pyridazinyl).

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl; each n1and n2 is, independently, an integer from 0 to 2 (e.g., n1 and n2 areboth 1 or n1 is 1 and n2 is 2); and R⁵ is selected from the groupconsisting of H, optionally substituted C₁₋₆ alkyl, and optionallysubstituted heterocyclyl (e.g., unsubstituted C₁₋₆ alkyl or C₁₋₆ alkylsubstituted with optionally substituted pyrrolyl, optionally substitutedimidazolyl, optionally substituted pyrazolyl, optionally substitutedpyridinyl, optionally substituted pyrazinyl, optionally substitutedpyrimidinyl, or optionally substituted pyridazinyl). In someembodiments, the compound is selected from the group consisting of L-2,L-5, L-6, L-22, L-23, L-24, L-25, L-26, L-28, L-29, L-45, and L-48, or apharmaceutically acceptable salt thereof.

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,optionally substituted C₁₁₋₂₄ alkyl, optionally substituted C₁₁₋₂₄alkenyl, optionally substituted C₁₁₋₂₄ alkynyl, optionally substitutedC₁₁₋₂₄ hetero alkyl, optionally substituted C₁₁₋₂₄ heteroalkenyl,optionally substituted C₁₁₋₂₄ heteroalkynyl; each n1 and n2 is,independently, an integer from 0 to 2 (e.g., n1 and n2 are both 1 or n1is 1 and n2 is 2); and R⁵ is selected from the group consisting of H,optionally substituted C₁₋₆ alkyl, and optionally substitutedheterocyclyl (e.g., unsubstituted C₁₋₆ alkyl or C₁₋₆ alkyl substitutedwith optionally substituted pyrrolyl, optionally substituted imidazolyl,optionally substituted pyrazolyl, optionally substituted pyridinyl,optionally substituted pyrazinyl, optionally substituted pyrimidinyl, oroptionally substituted pyridazinyl). In some embodiments, the compoundis selected from the group consisting of L-27 and L-47, or apharmaceutically acceptable salt thereof.

In some embodiments for any formula described herein (e.g., formulas(I), (IIa), and (IIb)), R⁵ is C₁₋₆ alkyl substituted with NR^(5a)R^(5b),where each R^(5a) and R^(5b) is, independently, H, optionallysubstituted C₁₋₆ alkyl (e.g., optionally substituted C₁₋₆ alkyl), andwhere R^(5a) and R^(5b) can combine together to form optionallysubstituted C₃₋₇ heterocyclyl. In some embodiments, R⁵ is optionallysubstituted heterocyclyl (e.g., optionally substituted pyrrolidinyl,optionally substituted imidazolidinyl, optionally substitutedpyrazolidinyl, optionally substituted piperidinyl, optionallysubstituted piperazinyl, optionally substituted azepanyl, optionallysubstituted pyrrolyl, optionally substituted imidazolyl, optionallysubstituted pyrazolyl, optionally substituted pyridinyl, optionallysubstituted pyrazinyl, optionally substituted pyrimidinyl, or optionallysubstituted pyridazinyl).

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,optionally substituted C₁₁₋₂₄ alkyl, optionally substituted C₁₁₋₂₄alkenyl, optionally substituted C₁₁₋₂₄ alkynyl, optionally substitutedC₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄ heteroalkenyl,optionally substituted C₁₁₋₂₄ heteroalkynyl; and each n1 and n2 is,independently, an integer from 0 to 2 (e.g., n1 and n2 are both 1 or n1is 1 and n2 is 2). In some embodiments, the compound is L-46, or apharmaceutically acceptable salt thereof.

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl; R³ is Hor optionally substituted C₁₋₆ alkyl; L¹ is optionally substituted C₁₋₆alkylene; and each R⁵ and R⁶ is, independently, H or optionallysubstituted C₁₋₆ alkyl, or where R⁵ and R⁶ combine to form an optionallysubstituted C₃₋₇ heterocyclyl.

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl; R³ is Hor optionally substituted C₁₋₆ alkyl; L¹ is optionally substituted C₁₋₆alkylene; and each R⁵ and R⁶ is, independently, H or optionallysubstituted C₁₋₆ alkyl, or where R⁵ and R⁶ combine to form an optionallysubstituted C₃₋₇ heterocyclyl.

In some embodiments of formulas (IId) or (IIe), R⁵ and R⁶ combine toform optionally substituted pyrrolidinyl, optionally substitutedimidazolidinyl, optionally substituted pyrazolidinyl, optionallysubstituted piperidinyl, optionally substituted piperazinyl, oroptionally substituted azepanyl.

In some embodiments, the compound is selected from the group consistingof L-1, L-3, L-4, L-7, L-9, L-10, L-11, L-12, L-15, L-16, L-17, L-18,L-19, L-30, L-31, L-32, L-33, L-34, L-42, L-43, and L-49, or apharmaceutically acceptable salt thereof.

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl; R³ is Hor optionally substituted C₁₋₆ alkyl; L¹ is optionally substituted C₁₋₆alkylene; each n3 and n4 is, independently, an integer from 0 to 2; andR⁵ is H or optionally substituted C₁₋₆ alkyl.

In some embodiments, the compound is selected from the group consistingof L-14, L-21, and L-36, or a pharmaceutically acceptable salt thereof.

In some embodiments of any formula described herein (e.g., formulas(IId)-(IIj), e.g., formulas (IId)-(IIg)), R³ is C₁₋₆ alkyl substitutedwith —NR^(3a)R^(3b) and where each R^(3a) and R^(3b) is, independently,H or optionally substituted C₁₋₆ alkyl. In some embodiments, R³ isunsubstituted C₁₋₆ alkyl.

In some embodiments of any formula described herein (e.g., formulas(IId)-(IIj), e.g., formulas (IId)-(IIg)), L¹ is C₁₋₆ alkylenesubstituted with methyl, ethyl, propyl, or —NR^(La)R^(Lb), where eachR^(La) and R^(Lb) is, independently, H or optionally substituted C₁₋₆alkyl.

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl; R³ is Hor optionally substituted C₁₋₆ alkyl; L¹ is optionally substituted C₁₋₆alkylene; and R⁵ is H or optionally substituted C₁₋₆ alkyl.

In some embodiments, L¹ is linked to the imidazolyl group at the4-position.

In some embodiments, the compound is selected from the group consistingof L-8, L-13, L-20, L-35, and L-44, or a pharmaceutically acceptablesalt thereof.

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl; R³ is Hor optionally substituted C₁₋₆ alkyl; L¹ is optionally substituted C₁₋₆alkylene; and each R⁵ and R⁶ is, independently, H or optionallysubstituted C₁₋₆ alkyl.

In some embodiments, the compound (e.g., lipid or cationic lipid) hasthe formula:

or a pharmaceutically acceptable salt thereof, where each R¹ and R² is,independently, optionally substituted C₁₁₋₂₄ alkyl, optionallysubstituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄ alkynyl,optionally substituted C₁₁₋₂₄ heteroalkyl, optionally substituted C₁₁₋₂₄heteroalkenyl, or optionally substituted C₁₁₋₂₄ heteroalkynyl; R³ is Hor optionally substituted C₁₋₆ alkyl; and each R⁵ and R⁶ is,independently, H or optionally substituted C₁₋₆ alkyl.

In some embodiments of any formula described herein (e.g., formulas(IId)-(IIk), e.g., formulas (IIi)-(IIk)), each R⁵ and R⁶ is,independently, C₁₋₆ alkyl substituted with —NR^(5a)R^(5b) and where eachR^(5a) and R^(5b) is, independently, H or optionally substituted C₁₋₆alkyl.

In some embodiments, the compound is selected from the group consistingof L-37, L-38, L-39, L-40, and L-41, or a pharmaceutically acceptablesalt thereof.

In some embodiments of any formula described herein (e.g., formulas(IId)-(IIk)), L¹ is optionally substituted C₁₋₆ alkylene.

In some embodiments of any formula described herein (e.g., formulas (I)or (IIa)-(IIk)), R³ is optionally substituted C₁₋₆ alkyl. In someembodiments, each R¹ and R² is, independently, unsubstituted C₁₁₋₂₄alkenyl or unsubstituted C₁₁₋₂₄ heteroalkenyl, including straight andbranched forms (e.g., each R¹ and R² is, independently, unsubstitutedC₁₁₋₂₄ alkenyl or unsubstituted C₁₁₋₂₄ heteroalkenyl containing one ormore double bonds). In some embodiments, one of R¹ or R² is notsaturated C₁₁₋₂₄ alkyl. In some embodiments, both R¹ and R² are notsaturated C₁₁₋₂₄ alkyl. In some embodiments, each R¹ and R² is,independently, selected from the group consisting of linolenyl (C18:3),linolenyloxy (C18:3), linolenoyl (C18:3), linoleyl (C18:2), linoleyloxy(C18:2), linoleoyl (C18:2), oleyl (C18:1), oleyloxy (18:1),oleyloxymethylene (18:1), oleoyl (C18:1), oleoylmethylene (C18:1),stearyl (C18:0), stearyloxy (C18:0), stearoyl (C18:0), palmityl (C16:0),palmityloxy (C16:0), palmitoyl (C16:0), palmitoylmethylene (C16:0),myristyl (C14:0), myristyloxy (C14:0), myristoyl (C14:0), lauryl(C12:0), lauryloxy (C12:0), and lauroyl (C12:0), e.g., linoleyl (C18:2)or oleyl (C18:1). In some embodiments, R¹ and R² are the same ordifferent.

In some embodiments of any formula described herein (e.g., formulas (I)or (IIa)-(IIk)), R³ or R⁴, but not both R³ and R⁴, is substituted with aprimary amine. In some embodiments, both R³ and R⁴ are not substitutedwith a primary amine.

In some embodiments of any formula described herein (e.g., formulas (I)or (IIa)-(IIk)), R³ and R⁴, together with the N to which they areattached, include a head group of one of H-1 to H-52 from Tables 2 and3. In some embodiments, each R¹ and R² is, independently, selected fromthe group consisting of linolenyl (C18:3), linolenyloxy (C18:3),linolenoyl (C18:3), linoleyl (C18:2), linoleyloxy (C18:2), linoleoyl(C18:2), oleyl (C18:1), oleyloxy (18:1), oleyloxymethylene (18:1),oleoyl (C18:1), oleoylmethylene (C18:1), stearyl (C18:0), stearyloxy(C18:0), stearoyl (C18:0), palmityl (C16:0), palmityloxy (C16:0),palmitoyl (C16:0), palmitoylmethylene (C16:0), myristyl (C14:0),myristyloxy (C14:0), myristoyl (C14:0), lauryl (C12:0), lauryloxy(C12:0), and lauroyl (C12:0), e.g., each R¹ and R² is, independently,linoleyl (C18:2) or oleyl (C18:1).

In another aspect, the compound of the invention include R¹R²—CH-A,where R¹ and R² is a tail group (e.g., any described herein, e.g., inTable 4) and A is a head group (e.g., any described herein, e.g., inTables 2 and 3). In some embodiments, the head group is one of H-1 toH-52, e.g., H-2, H-5, H-6, H-19, H-26, or H-43 (e.g., H-5 or H-43).

In another aspect, the compound of the invention is any compoundprovided in Table 1, or a pharmaceutically acceptable salt thereof.

In one aspect, the invention features a formulation including anycompound described herein (e.g., one or more compound provided in Table1), or a pharmaceutically acceptable salt thereof.

In some embodiments, the formulation includes two or more of thecompounds, e.g., two, three, four, five, six, seven, or more of thecompounds.

In any of the above aspects, the compounds of the invention includes twounsaturated lipid tail groups (e.g., each R¹ and R² is, independently,optionally substituted C₁₁₋₂₄ alkenyl, optionally substituted C₁₁₋₂₄alkynyl, optionally substituted C₁₁₋₂₄ heteroalkenyl, or optionallysubstituted C₁₁₋₂₄ heteroalkynyl).

In any of the above aspects, the compounds of the invention includelipid tail groups, where these groups do not include an oxygen adjacentto —CHR³R⁴ (e.g., each R¹ and R² is, independently, optionallysubstituted C₁₁₋₂₄ alkyl, optionally substituted C₁₁₋₂₄ alkenyl, oroptionally substituted C₁₁₋₂₄ alkynyl).

In any of the above aspects, the compounds of the invention includelipid tail groups, where these groups do not include one or morebiodegradable groups (e.g., one or more ester groups).

In any of the above aspects, the compounds of the invention includes twolipid tail groups having more than 11, 12, 13, 14, 15, 16, or 18 carbons(e.g., each R¹ and R² is, independently, optionally substituted C₁₇₋₂₄alkenyl, optionally substituted C₁₅₋₂₄ alkynyl, optionally substitutedC₁₅₋₂₄ heteroalkenyl, or optionally substituted C₁₅₋₂₄ heteroalkynyl;each R¹ and R² is, independently, optionally substituted C₁₆₋₂₄ alkenyl,optionally substituted C₁₆₋₂₄ alkynyl, optionally substituted C₁₆₋₂₄heteroalkenyl, or optionally substituted C₁₆₋₂₄ heteroalkynyl; each R¹and R² is, independently, optionally substituted C₁₇₋₂₄ alkenyl,optionally substituted C₁₇₋₂₄ alkynyl, optionally substituted C₁₇₋₂₄heteroalkenyl, or optionally substituted C₁₇₋₂₄ heteroalkynyl; or eachR¹ and R² is, independently, optionally substituted C₁₈₋₂₄ alkenyl,optionally substituted C₁₈₋₂₄ alkynyl, optionally substituted C₁₈₋₂₄heteroalkenyl, or optionally substituted C₁₈₋₂₄ heteroalkynyl).

In any of the above aspects, the compounds of the invention do notcontain a urea group (e.g., neither R³ nor R⁴ is an optionallysubstituted amide). In some embodiments, the compounds do not contain acarbamyl group. In some embodiments, the compounds do not contain morethan one primary amine group (e.g., do not contain two primary aminegroups or do not contain any primary amine groups in one or more ofR¹-R⁶, e.g., in either R³ or R⁴). In particular embodiments, thecompounds include only one primary amine or no primary amines (e.g.,only one primary amine or no primary amines are present in one or moreof R¹-R⁶, e.g., in either R³ or R⁴).

In any of the above aspects, the compounds of the invention do notcontain a hydroxy group (e.g., neither R¹ nor R² is substituted withone, two, or three hydroxy groups; or neither R³ nor R⁴ is substitutedwith one, two, or three hydroxy groups). In some embodiments, when R¹ orR² is a saturated C₁₁₋₂₄ alkyl group (e.g., a saturated C₁₅ alkyl, asaturated C₁₆ alkyl, a saturated C₁₇ alkyl, or a saturated C₁₈ alkyl),R¹ and/or R² is not substituted with one, two, or three hydroxy groups.In some embodiments, when R¹ or R² is a saturated C₁₁₋₂₄ alkyl group(e.g., a saturated C₁₅ alkyl, a saturated C₁₆ alkyl, a saturated C₁₇alkyl, or a saturated C₁₈ alkyl), R³ and/or R⁴ is not substituted withone, two, or three hydroxy groups.

In any of the above aspects, the compounds of the invention include nomore than two amide groups (e.g., no more than two or one amide groupsin the head group of the compound). In other embodiments, the compoundsinclude zero, one, or two amide groups in one or more of R¹-R⁶ (e.g.,zero, one, or two amide groups in R³ or R⁴). In yet other embodiments,the compounds can include one, and only one, amide group (e.g., caninclude one, and only one, amide groups in R³ or R⁴). In furtherembodiments, the compounds include one, and only, amide group or noamide groups (e.g., include one, and only one, amide group or no amidegroups in R³ or R⁴).

In any of the above aspects, the compounds of the invention excludeN-(4-N′,N′-dimethylamino)butanoyl-(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-amine orN-(3-N′,N′-dimethylamino)propanoyl-(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-amine,or salts thereof. In some embodiments, the compounds of the inventionexclude N-methyl-N-(4-N′,N′-dimethylamino)butanoyl-(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-amine orN-methyl-N-(3-N′,N′-dimethylamino)propanoyl-(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-amine,or salts thereof.

In any of the above aspects, the compounds of the invention excludeN-(4-N′,N′-dimethylamino)butanoyl-(6Z,9Z,28Z)-heptatriaconta-6,9,28-trien-19-amine,N-methyl-N-(4-N′,N′-dimethylamino)butanoyl-(6Z,9Z,28Z)-heptatriaconta-6,9,28-trien-19-amine,N-(4-N′,N′-dimethylamino)butanoyl-(6Z,9Z,28Z,31Z,34Z)-heptatriaconta-6,9,28,31,34-pentaen-19-amine,N-methyl-N-(4-N′,N′-dimethylamino)butanoyl-(6Z,9Z,28Z,31Z,34Z)-heptatriaconta-6,9,28,31,34-pentaen-19-amine,N-(3-N′,N′-dimethylamino)propanoyl-(6Z,9Z,28Z)-heptatriaconta-6,9,28-trien-19-amine,N-methyl-N-(3-N′,N′-dimethylamino)propanoyl-(6Z,9Z,28Z)-heptatriaconta-6,9,28-trien-19-amine,N-(3-N′,N′-dimethylamino)propanoyl-(6Z,9Z,28Z,31Z,34Z)-heptatriaconta-6,9,28,31,34-pentaen-19-amine,N-methyl-N-(3-N′,N′-dimethylamino)propanoyl-(6Z,9Z,28Z,31Z,34Z)-heptatriaconta-6,9,28,31,34-pentaen-19-amine,or salts thereof.

In any of the above aspects, the compounds of the invention excludedi((Z)-non-2-en-1-yl)9-((3-(dimethylamino)propanoyl)amino)heptadecanedioate,di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)amino)heptadecanedioate,di((Z)-non-2-en-1-yl)9-((5-(dimethylamino)pentanoyl)amino)heptadecanedioate, or saltsthereof.

In any of the above aspects, the compounds of the invention has a pKavalue less than 6.2 and more than 6.5 (e.g., a pKa value between 4.0 and6.2, such as between 4.0 and 5.2, between 4.0 and 5.6, or between 4.0and 5.8; or between 6.5 and 8.5, e.g., between 6.5 and 7.0, between 6.5and 7.5, or between 6.5 and 8.0). In particular embodiments, the pKavalue is between about 5.0 and about 6.0 (e.g., between 5.0 and 5.5,between 5.0 and 5.6, between 5.0 and 5.7, between 5.0 and 5.8, between5.0 and 5.9, between 5.0 and 6.0, between 5.2 and 5.5, between 5.2 and5.6, between 5.2 and 5.7, between 5.2 and 5.8, between 5.2 and 5.9,between 5.2 and 6.0, between 5.4 and 5.5, between 5.4 and 5.6, between5.4 and 5.7, between 5.4 and 5.8, between 5.4 and 5.9, between 5.4 and6.0, between 5.6 and 5.7, between 5.6 and 5.8, between 5.6 and 5.9, orbetween 5.6 and 6.0). The pKa value can be determined by any usefulmethod, e.g., measuring fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS), zeta potential measurements, etc. In particularembodiments, the pKa value is the ratio of the concentration of chargedcationic lipid and the concentration of uncharged lipid (e.g., asmeasured by in situ TNS fluorescence titration, where pKa is defined asthe pH at half-maximal fluorescence intensity).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary manufacturing process for an anionicagent-comprising particle of the instant invention. Performance of theprocess involves initially combining a lipid complex suspended in anacidic aqueous solution (here, 60 mM HCl at pH 2.3) and anionic agent(here, RNA dissolved in water), diluting such solution with water, thenadding a lipidic solution dissolved in a solvent (here, ethanol) to thecomplex-anionic agent mixture, thereby producing particles comprisingthe anionic agent. Particles thus formed are then diluted in anadditional volume of water and are optionally then subjected tofiltration (here, tangential flow filtration (TFF)) to remove solventand concentrate the particles prior to use.

FIG. 2 shows sizing results for particles formulated by three distinctmethods: particles formulated by a process that involved adding aqueouslipid-DsiRNA complexes into additional lipids dissolved in ethanol(“2072 process”, variation 1, top panel); particles formulated by aprocess that involved adding additional lipids dissolved in ethanol intoaqueous lipid-DsiRNA complexes (“2072 process”, variation 2, middlepanel); and particles formulated by a process that incorporatedidentical total amounts and proportions of components as “2072” but thatfeatured a step that allowed for concentration of lipids within ethanol,specifically by dissolving a number of lipids in ethanol prior todissolution of cholesterol in the lipid-containing ethanol solution suchprocess allowed for remarkable concentration of such lipids within suchethanol solution (“2141 process”, lower panel).

FIG. 3 shows the result of particle sizing experiments performed upon“2072” (top) and “2141” particle populations after initial performanceof size-exclusion chromatography (“SEC”) and selection of fractions 2-5.

FIG. 4 shows percent volume particle size assay results for particlesproduced by the “2072” (top) and “2141” (bottom) processes.

FIG. 5 shows in vivo target-specific knockdown results observed forparticles harboring an HPRT1-targeting DsiRNA which were formulated byvarious indicated processes, including “2072” and “2141”-relatedprocesses “2141”, “2137” and “2144”. HPRT1 raw data was normalized tohSFRS9 levels and then plotted.

FIG. 6 demonstrates the in vivo efficacy and tolerability profiles of“2072”-produced particles and “2141”-produced particles, each harboringa MYC-targeting DsiRNA payload as indicated.

FIG. 7 demonstrates gross in vivo tolerabilities (body weights and liverweights) of “2072”-produced particles and “2141”-produced particles.

FIG. 8 shows the results of toxicity marker assessments for both“2072”-produced particles and “2141”-produced particles.

FIG. 9 shows results of efficacy testing for Example 6.

DETAILED DESCRIPTION

The present invention is directed to processes for formulation ofanionic agents, performance of which enhance the probability that suchanionic agents achieve intracellular localization upon administration tomammalian cells and/or mammals.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

As used herein, the term “about” means±10% of the recited value.

As used herein, the term “acidic aqueous solution” is intended to meanan aqueous solution of pH 1.0 to pH 6.9, preferably of pH 2.0 to pH 4.0,which has a molarity of 5 to 200 mM, optionally 20 to 100 mM or 40 to 80mM. The acidic aqueous solution may be selected from aqueous solutionsof hydrochloric acid, citric acid, acetic acid and other acids. The typeand pH of acidic aqueous solution will vary depending on the type oflipid and/or anionic agent to be suspended or dissolved in suchsolution.

By “alkenyl” is meant a monovalent straight or branched chain group of,unless otherwise specified, from 2 to 24 carbon atoms containing one ormore carbon-carbon double bonds. Alkenyl groups are exemplified byethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl,2-butenyl, oleyl, linoleyl, linolenyl, and the like. The term “C_(x-y)alkenyl” represents alkenyl groups having between x and y carbons.Exemplary values for x are 2, 3, 4, 5, and 11; for y are 3, 4, 5, 6, and24; and for x to y are 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5,2 to 4, 10 to 24, 11 to 24, 12 to 24, 14 to 24, 16 to 24, 18 to 24, 10to 22, 11 to 22, 12 to 22, 14 to 22, 16 to 22, 18 to 22, 10 to 20, 11 to20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20. In some embodiments, thealkenyl can be further substituted with 1, 2, 3, or 4 substituent groupsas defined herein for an alkyl group.

By “alkyl” is meant a monovalent straight or branched saturated groupof, unless otherwise specified, 1 to 24 carbon atoms. Alkyl groups areexemplified by methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,iso-butyl, tert-butyl, neopentyl, lauryl, myristyl, palmityl, stearyl,and the like, and may be optionally substituted with one, two, three,or, in the case of alkyl groups of two carbons or more, foursubstituents independently selected from the group consisting of: (1)alkoxy; (2) amino, as defined herein; (3) halo, such as F, Cl, Br, or I;(4) (heterocyclyl)oxy; (5) heterocyclyl; (6) alkyl; (7) alkenyl; (9)alkynyl; (10) cycloalkyl; (11) hydroxy; (12) nitro; or (13) oxo (e.g.,carboxyaldehyde or acyl). In some embodiments, each of these groups canbe further substituted as described herein. The term “C_(x-y) alkyl”represents alkyl groups having between x and y carbons. Exemplary valuesfor x are 1, 2, 3, 4, 5, and 11; for y are 2, 3, 4, 5, 6, and 24; andfor x to y are 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4,10 to 24, 11 to 24, 12 to 24, 14 to 24, 16 to 24, 18 to 24, 10 to 22, 11to 22, 12 to 22, 14 to 22, 16 to 22, 18 to 22, 10 to 20, 11 to 20, 12 to20, 14 to 20, 16 to 20, or 18 to 20.

The term “alkylene” and the prefix “alk-,” as used herein, represent apolyvalent (e.g., divalent) hydrocarbon group derived from a straight orbranched chain hydrocarbon by the removal of two hydrogen atoms.Alkylene groups are exemplified by methylene, ethylene, isopropylene,and the like. The term “C_(x-y) alkylene” represent alkylene groupshaving between x and y carbons. Exemplary values for x are 1, 2, 3, 4,and 5, and exemplary values for y are 2, 3, 4, 5, and 6. In someembodiments, the alkylene can be further substituted with 1, 2, 3, or 4substituent groups as defined herein for an alkyl group.

By “alkynyl” is meant a monovalent straight or branched chain group of,unless otherwise specified, from 2 to 24 carbon atoms containing one ormore carbon-carbon triple bonds. Alkynyl groups are exemplified byethynyl, 1-propynyl, and the like. The term “C_(x-y) alkynyl” representsalkynyl groups having between x and y carbons. Exemplary values for xare 2, 3, 4, 5, and 11; for y are 3, 4, 5, 6, and 24; and for x to y are2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 10 to 24, 11 to24, 12 to 24, 14 to 24, 16 to 24, 18 to 24, 10 to 22, 11 to 22, 12 to22, 14 to 22, 16 to 22, 18 to 22, 10 to 20, 11 to 20, 12 to 20, 14 to20, 16 to 20, or 18 to 20. In some embodiments, the alkynyl can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein for an alkyl group.

By “amide” is meant an amine group, as defined herein, attached to theparent molecular group through a carbonyl group.

By “amino,” as used herein, is meant —N(R^(N1))₂, wherein each R^(N1)is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, heterocyclyl (e.g., heteroaryl),alkheterocyclyl (e.g., alkheteroaryl), or two R^(N1) combine to form aheterocyclyl or an N-protecting group, and wherein each R^(N2) is,independently, H, alkyl, or aryl. In a preferred embodiment, amino is—NH₂, or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and eachR^(N2) can be H, alkyl, or aryl. By “primary amine” is meant a grouphaving the structure —NH₂.

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group.

By “amount sufficient” of an agent is meant the amount of the agentsufficient to effect beneficial or desired results, such as clinicalresults, and, as such, an amount sufficient depends upon the context inwhich it is applied. For example, in the context of administering aformulation that reduces the expression level of a target gene, theamount sufficient of the formulation is an amount sufficient to achievea reduction in the expression level of the target gene as compared tothe response obtained without administration of the formulation.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids. Representative examples of phospholipidsinclude, but are not limited to, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Othercompounds lacking in phosphorus, such as sphingolipid, glycosphingolipidfamilies, diacylglycerols, and β-acyloxyacids, are also within the groupdesignated as amphipathic lipids. Additionally, the amphipathic lipidsdescribed above can be mixed with other lipids including triglyceridesand sterols.

As used herein, the term “anionic agent” refers to a chemical moietycomprising at least one negatively charged atom, which optionally may beincorporated into a formulation (e.g., as a payload). By “polyanionicpayload” is meant a chemical moiety comprising multiple negativelycharged atoms that may be incorporated into a formulation. Examples of apolyanionic payload include nucleic acids, RNAi agents, siRNA, dsRNA,miRNA, shRNA, DsiRNA, and antisense payloads.

By “anionic lipid” is meant any lipid molecule that has a net negativecharge at physiological pH. These lipids include, but are not limitedto, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

As used herein, the term “antisense compound” or “antisense payload”encompasses, inter alia, single-stranded antisense oligonucleotides(DNA, DNA-like, RNA, RNA-like) or certain double-stranded orself-hybridizing constructs comprising an antisense orientationoligonucleotide, antisense PNAs, ribozymes and external guide sequences(sequences that recruit RNase P, as described, e.g., in Guerrier-Takadaet al., Proc. Natl. Acad. Sci. USA 94:8468, 1997). Antisense compoundscan exert their effect by a variety of means. One such means is theantisense-mediated direction of an endogenous nuclease, such as RNase Hin eukaryotes or RNase P in prokaryotes (Chiang et al., J. Biol. Chem.1266:18162, 1991; Forster et al., Science, 249:783, 1990).

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

The term “cancer” refers to any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,liver cancer, lung cancer, colon cancer, rectal cancer, anal cancer,bile duct cancer, small intestine cancer, stomach (gastric) cancer,esophageal cancer; gallbladder cancer, pancreatic cancer, appendixcancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer,renal cancer (e.g., renal cell carcinoma), cancer of the central nervoussystem, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head andneck cancers, osteogenic sarcomas, and blood cancers. Non-limitingexamples of specific types of liver cancer include hepatocellularcarcinoma (HCC), secondary liver cancer (e.g., caused by metastasis ofsome other non-liver cancer cell type), and hepatoblastoma. As usedherein, a “tumor” comprises one or more cancerous cells.

By “cationic lipid” is meant any lipid molecule that has a net positivecharge at physiological pH. Exemplary cationic lipids include anydescribed herein, e.g., in Table 1. In certain embodiments, the cationiclipid may comprise from about 20 mol % to about 50 mol % or about 40 mol% of the total lipid present in the particle.

As used herein, the term “carbamyl” refers to a carbamate group havingthe structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning ofeach R^(N1) is found in the definition of “amino” provided herein, and Ris alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

By “cycloalkyl” is meant a monovalent saturated or partially unsaturated3- to 10-membered monocyclic or polycyclic (e.g., bicyclic or tricyclic)hydrocarbon ring system. Examples include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and cycloheptyl.

By “Dicer-substrate RNA” or “DsiRNA” is meant a class of 25+, e.g.,25-35 (e.g., 25-27, such as double stranded regions of 25 nucleotides inlength) nucleotide double-stranded molecules that are capable of genesilencing. Due to its longer length compared to other RNAi agents,DsiRNA are likely substrates of Dicer.

By “double-stranded molecule” is meant a double-stranded RNA:RNA orRNA:DNA molecule that can be used to silence a gene product through RNAinterference.

By “expression” is meant the detection of a gene or polypeptide bymethods known in the art. For example, DNA expression is often detectedby Southern blotting or polymerase chain reaction (PCR), and RNAexpression is often detected by Northern blotting, RT-PCR, gene arraytechnology, or RNAse protection assays. Methods to measure proteinexpression level generally include, but are not limited to, Westernblotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surfaceplasmon resonance, chemiluminescence, fluorescent polarization,phosphorescence, immunohistochemical analysis, matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,microcytometry, microscopy, fluorescence activated cell sorting (FACS),and flow cytometry, as well as assays based on a property of the proteinincluding, but not limited to, enzymatic activity or interaction withother protein partners.

The term “fusogenic” refers to the ability of a lipid particle, such asthose described herein, to fuse with the membranes of a cell. Themembranes can be either the plasma membrane or membranes surroundingorganelles, e.g., endosome, nucleus, etc.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

By “heteroalkenyl” is meant an alkenyl group, as defined herein, inwhich one or more of the constituent carbon atoms have each beenreplaced by O, N, or S. Exemplary heteroalkenyl groups include alkenylgroups, as described herein, substituted with an oxo group and/orattached to the parent molecular group through an oxygen atom. In someembodiments, the heteroalkenyl group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkyl groups.

By “heteroalkyl” is meant an alkyl group, as defined herein, in whichone or more of the constituent carbon atoms have each been replaced byO, N, or S. Exemplary heteroalkyl groups include alkyl groups, asdescribed herein, substituted with an oxo group and/or attached to theparent molecular group through an oxygen atom. In some embodiments, theheteroalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which 1 or 2 of the constituent carbon atoms haveeach been replaced by 0, N, or S. In some embodiments, theheteroalkylene group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkylene groups. The term“C_(x-y) heteroalkylene” represent heteroalkylene groups having betweenx and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 11; for yare 2, 3, 4, 5, 6, and 24; and for x to y are 1 to 10, 1 to 9, 1 to 8, 1to 7, 1 to 6, 1 to 5, 1 to 4, 10 to 24, 11 to 24, 12 to 24, 14 to 24, 16to 24, 18 to 24, 10 to 22, 11 to 22, 12 to 22, 14 to 22, 16 to 22, 18 to22, 10 to 20, 11 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20.

By “heteroalkynyl” is meant an alkynyl group, as defined herein, inwhich one or more of the constituent carbon atoms have each beenreplaced by O, N, or S. Exemplary heteroalkynyl groups include alkynylgroups, as described herein, substituted with an oxo group and/orattached to the parent molecular group through an oxygen atom. In someembodiments, the heteroalkynyl group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkyl groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 3-, 4-, 5-, 6-, 7-,or 8-membered ring, unless otherwise specified, containing one, two,three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. The heterocyclyl may besaturated or unsaturated and contain between 0 and 3 unsaturated bonds.For example, the 5-membered ring has zero to two double bonds, and the6- and 7-membered rings have zero to three double bonds. Certainheterocyclyl groups include from 2 to 9 carbon atoms, e.g., from 3 to 7carbon atoms. Other such groups may include up to 12 carbon atoms. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. Examples of heterocyclic groups includeaziridinyl, azetidinyl, pyrrolinyl, pyrrolyl, pyrrolidinyl, pyrazolyl,pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl,pyridyl, pyrimidinyl, piperidinyl, azepanyl, pyrazinyl, piperazinyl,diazepanyl, morpholinyl, tetrahydrofuranyl, dihydrofuranyl, and thelike.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

By “hybridize” is meant to pair to form a double-stranded moleculebetween sufficiently complementary polynucleotides, as defined herein,or portions thereof, under various conditions of stringency. (See, e.g.,Wahl et al., Methods Enzymol. 152:399 (1987); Kimmel, Methods Enzymol.152:507 (1987)). For example, high stringency salt concentration willordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate,less than about 500 mM NaCl and 50 mM trisodium citrate, or less thanabout 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide or at least about 50%formamide. High stringency temperature conditions will ordinarilyinclude temperatures of at least about 30° C., 37° C., or 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In one embodiment, hybridization will occur at 30°C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In analternative embodiment, hybridization will occur at 50° C. or 70° C. in400 mM NaCl, 40 mM PIPES, and 1 mM EDTA, at pH 6.4, after hybridizationfor 12-16 hours, followed by washing. Additional preferred hybridizationconditions include hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC,50% formamide followed by washing at 70° C. in 0.3×SSC or hybridizationat 70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washingat 67° C. in 1×SSC. Useful variations on these conditions will bereadily apparent to those skilled in the art. One such exemplaryvariation includes assessment of hybridization under conditions designedto mimic physiological intracellular conditions, wherein cations andanions are assorted in the following proportions: for cations,Sodium:Potassium:Calcium:Magnesium at 10:160:2:26; and for anions,Chloride:Bicarbonate:Phosphate:Sulfate:Gluconate at 3:10:100:20:65.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “hydroxy,” as used herein, represents an —OH group.

The term “lipid” refers to any fatty acid derivative which is capable offorming a micelle such that a hydrophobic portion of the lipid materialis shielded from an aqueous phase/solution by a hydrophilic portion thatorients toward the aqueous phase, or is capable of forming a bilayersuch that a hydrophobic portion of the lipid material orients toward thebilayer while a hydrophilic portion orients toward the aqueous phase.Hydrophilic characteristics derive from the presence of phosphato,carboxylic, sulfato, amino, sulfhydryl, nitro, and other like groups.Hydrophobicity could be conferred by the inclusion of groups thatinclude, but are not limited to, long chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic or heterocyclic group(s). Preferred lipids arephosphoglycerides and sphingolipids, representative examples of whichinclude phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidic acid,palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine ordilinoleoylphosphatidylcholine could be used. Other compounds lacking inphosphorus, such as sphingolipid and glycosphingolipid families are alsowithin the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

The term “lipid conjugate” refers to a conjugated lipid, optionally onethat inhibits aggregation of lipid particles. Such lipid conjugatesinclude, but are not limited to, PEG-lipid conjugates such as, e.g., PEGcoupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see,e.g., U.S. Provisional Application No. 61/294,828, filed Jan. 13, 2010,and U.S. Provisional Application No. 61/295,140, filed Jan. 14, 2010),polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Additional examples of POZ-lipid conjugates are described in PCTPublication No. WO 2010/006282. PEG or POZ can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester containing linker moieties andester-containing linker moieties. In certain embodiments, non-estercontaining linker moieties, such as amides or carbamates, are used. Thedisclosures of each of the above patent documents are hereinincorporated by reference in their entirety for all purposes. Aconjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), aPEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or aPEG-distearyloxypropyl (Ci8). In certain embodiments, a conjugated lipidthat prevents aggregation of particles may be from 0 mol % to about 20mol % or about 2 mol % of the total lipid present in the particle.

By “lipid vector” is meant a liposome, lipoplex, micelle, lipidnanoparticle, core-based particle, particle comprising an RNA bindingagent-RNA aggregate which is combined with transfection lipid(s) orvesicle-based particle made by a process of the invention.

By “linker” is meant an optionally substituted polyvalent (e.g.,divalent) group containing one or more atoms. Examples of linkersinclude optionally substituted alkylene and heteroalkylene groups, asdescribed herein.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as an interfering RNA (e.g., DsiRNA) directly to a target sitewithin an organism. For example, an agent can be locally delivered bydirect injection into a disease site such as a tumor or other targetsite such as a site of inflammation or a target organ such as the liver,heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

By “microRNA” (miRNA) is meant a single-stranded RNA molecule that canbe used to silence a gene product through RNA interference.

The term “modified lipid” refers to lipids modified to aid in, forexample, inhibiting aggregation and/or precipitation, inhibiting immuneresponse and/or improving half-life in circulation in vivo. In certainaspects of the present invention, the modified lipids are neutrallipids. Modified neutral lipids include, but are not limited to,pegylated lipids, such as polyethyleneglycol 2000distearoylphosphatidylethanolamine (PEG(2000) DSPE); PEG-DMG; PEG-DMPE;PEG-DPPE; PEG-DPG; PEG-DOPE; or PEG-DOG.

As used herein, a “modified lipid which prevents particle aggregationduring lipid-anionic agent particle formation” is any modified lipidthat provides a means for increasing circulation lifetime and/orincreasing the delivery of the anionic agent-lipid particles to a targettissue. Exemplary such modified lipids include polyethylene glycol(PEG), PEG-ceramide, or ganglioside (e.g., GM1)-modified lipids.Typically, the concentration of the PEG, PEG-ceramide organglioside-modified lipids in the particle will be about 1-15%.

By “modulate” is meant that the expression of a gene, or level of an RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up-regulated or down-regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term modulate can include inhibition orgene silencing, and the level of expression of a gene or the level of anRNA molecule, or an equivalent thereof, is reduced by at least 10%(e.g., 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%), as compared to a control.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

The term “oxo” as used herein, represents ═O.

The term “urea” refers to a group having the structureNR^(N1)C(═O)NR^(N1), where the meaning of each R^(N1) is found in thedefinition of “amino” provided herein.

By “neutral lipid” is meant any of a number of lipid species that existeither in an uncharged or neutral zwitterionic form at a selected pH. Atphysiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid. The non-cationic lipid may bean anionic lipid or a neutral lipid including, but not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. In certain embodiments, the non-cationic lipid may befrom about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol %if cholesterol is included, of the total lipid present in the particle.In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

By “pharmaceutical composition” is meant a composition containing acompound described herein formulated with a pharmaceutically acceptableexcipient, and manufactured or sold with the approval of a governmentalregulatory agency as part of a therapeutic regimen for the treatment ofdisease in a mammal. Pharmaceutical compositions can be formulated, forexample, for oral administration in unit dosage form (e.g., a tablet,capsule, caplet, gelcap, or syrup); for topical administration (e.g., asa cream, gel, lotion, or ointment); for intravenous administration(e.g., as a sterile solution free of particulate emboli and in a solventsystem suitable for intravenous use); or in any other formulationdescribed herein.

By “pharmaceutically acceptable excipient” is meant any ingredient otherthan the compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being nontoxic and non-inflammatory in a patient. Excipients mayinclude, for example: antiadherents, antioxidants, binders, coatings,compression aids, disintegrants, dyes (colors), emollients, emulsifiers,fillers (diluents), film formers or coatings, flavors, fragrances,glidants (flow enhancers), lubricants, preservatives, printing inks,sorbents, suspensing or dispersing agents, sweeteners, and waters ofhydration. Exemplary excipients include, but are not limited to:butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate(dibasic), calcium stearate, croscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose,gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose,lactose, magnesium stearate, maltitol, mannitol, methionine,methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

By “pharmaceutically acceptable salt” is meant those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and animals without undue toxicity,irritation, allergic response and the like and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, pharmaceutically acceptable saltsare described in: Berge et al., J. Pharm. Sci. 66(1):1, 1977 and inPharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008. The salts can be prepared in situduring the final isolation and purification of the compounds of theinvention or separately by reacting the free base group with a suitableorganic acid. Representative acid addition salts include acetate,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,and the like.

By “RNA-binding agent” is meant any agent or combination of agentscapable of binding or hybridizing a nucleic acid, e.g., a nucleic acidpayload of a therapeutic formulation. RNA-binding agents include anylipid described herein (e.g., one or more cationic lipids, combinationsof one or more cationic lipids, such as those described herein or inTable 1, as well as combinations of one or more cationic lipids and anyother lipid, such as neutral lipids or PEG-lipid conjugates). TheRNA-binding agent can form any useful structure within a formulation,such as an internal aggregate.

By “RNAi agent” is meant any agent or compound that exerts a genesilencing effect by hybridizing a target nucleic acid. RNAi agentsinclude any nucleic acid molecules that are capable of mediatingsequence-specific RNAi (e.g., under stringent conditions), for example,a short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA(miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide,short interfering nucleic acid, short interfering modifiedoligonucleotide, chemically-modified siRNA, post-transcriptional genesilencing RNA (ptgsRNA), and Dicer-substrate RNA (DsiRNA).

By “short hairpin RNA” or “shRNA” is meant a sequence of RNA that makesa tight hairpin turn and is capable of gene silencing.

By “sense region” is meant a nucleotide sequence of a nucleic acid ofthe invention having sufficient complementarity to an antisense regionof another nucleic acid. In addition, the sense region of a nucleic acidof the invention can include a nucleotide sequence having homology witha target gene nucleotide sequence. By “antisense region” is meant anucleotide sequence of a nucleic acid of the invention having sufficientcomplementarity to a target gene nucleotide sequence.

“Serum-stable” in relation to nucleic acid-lipid particles such as thosedescribed herein means that the particle is not significantly degradedafter exposure to a serum or nuclease assay that would significantlydegrade free DNA or RNA. Suitable assays include, for example, astandard serum assay, a DNAse assay, or an RNAse assay.

By “silencing” or “gene silencing” is meant that the expression of agene or the level of an RNA molecule that encodes one or more proteinsis reduced in the presence of an RNAi agent below that observed undercontrol conditions (e.g., in the absence of the RNAi agent or in thepresence of an inactive or attenuated molecule such as an RNAi moleculewith a scrambled sequence or with mismatches). Gene silencing maydecrease gene product expression by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% (i.e., complete inhibition).

By “small inhibitory RNA,” “short interfering RNA,” or “siRNA” is meanta class of 10-40 (e.g., 15-25, such as 21, or 25-35 and/or 25-30, suchas 25, 26 or 27) nucleotide double-stranded molecules that are capableof gene silencing. Most notably, siRNA are typically involved in the RNAinterference (RNAi) pathway by which the siRNA interferes with theexpression of a specific gene product.

The term “solubility” refers to the quantity of a compound (the solute)that dissolves in a given quantity of solvent to form a saturatedsolution. A “solution” refers to a homogeneous mixture of a liquid (thesolvent) with a gas or solid (the solute). In a solution the moleculesof the solute are discrete and mixed with the molecules of the solvent.The solubility of a substance depends on the temperature. The“solubility in water” refers to the solubility of a solute in thesolvent water.

By “subject” is meant either a human or non-human animal (e.g., amammal).

By “substantial identity” or “substantially identical” is meant apolypeptide or polynucleotide sequence that has the same polypeptide orpolynucleotide sequence, respectively, as a reference sequence, or has aspecified percentage of amino acid residues or nucleotides,respectively, that are the same at the corresponding location within areference sequence when the two sequences are optimally aligned. Forexample, an amino acid sequence that is “substantially identical” to areference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identity to the reference amino acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 contiguous amino acids, more preferably at least 25, 50, 75,90, 100, 150, 200, 250, 300, or 350 contiguous amino acids, and mostpreferably the full-length amino acid sequence. For nucleic acids, thelength of comparison sequences will generally be at least 5 contiguousnucleotides, preferably at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 contiguous nucleotides, and most preferablythe full-length nucleotide sequence. Sequence identity may be measuredusing sequence analysis software on the default setting (e.g., SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705). Such software may match similar sequences by assigning degreesof homology to various substitutions, deletions, and othermodifications.

By “sufficiently complementary” is meant a polynucleotide sequence thathas the exact complementary polynucleotide sequence, as a target nucleicacid, or has a specified percentage or nucleotides that are the exactcomplement at the corresponding location within the target nucleic acidwhen the two sequences are optimally aligned. For example, apolynucleotide sequence that is “substantially complementary” to atarget nucleic acid sequence has at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the targetnucleic acid sequence. For RNAi agents having a length between 10 to 40nucleotides, sufficiently complementary sequences include those havingone, two, three, four, or five non-complementary nucleotides. Indeed, incertain embodiments that include, e.g., DsiRNA agents, an activedouble-stranded RNAi agent can possess as few as 15 to 19 consecutivenucleotides of guide strand which are sufficiently complementary to atarget nucleic acid, while there is no requirement for the remainder ofthe guide strand to possess any extent of complementarity with thetarget nucleic acid (though in certain embodiments, the remainder of theguide strand may partially or fully complementary with the nucleic acid(e.g., mRNA) that is targeted).

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas an interfering RNA (e.g., DsiRNA) within an organism. Some techniquesof administration can lead to the systemic delivery of certain agents,but not others. Systemic delivery means that a useful, preferablytherapeutic, amount of an agent is exposed to most parts of the body. Toobtain broad biodistribution generally requires a blood lifetime suchthat the agent is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site distal to the site of administration. Systemicdelivery of lipid particles can be by any means known in the artincluding, for example, intravenous, subcutaneous, and intraperitoneal.In a preferred embodiment, systemic delivery of lipid particles is byintravenous delivery.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA. In certain embodiments, the target nucleic acid is atarget mRNA.

By “transfection lipid” is meant any lipid or combination of lipidscapable of delivering a nucleic acid, e.g., a nucleic acid payload(optionally, the nucleic acid payload is in associated with an RNAbinding agent, e.g., one or more cationic lipids) Transfection lipidsinclude any lipid described herein (e.g., one or more cationic lipids,combinations of one or more cationic lipids, such as those describedherein or in Table 1, as well as combinations of one or more cationiclipids and any other lipid or agent, such as neutral lipids, anioniclipids, PEG-lipid conjugates, or sterol derivatives). The transfectionlipid or combinations including such a transfection lipid can form anyuseful structure within a formulation, such as an external, aggregatesurface.

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, such as clinicalresults. Beneficial or desired results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions;diminishment of extent of disease, disorder, or condition; stabilization(i.e., not worsening) of a state of disease, disorder, or condition;prevention of spread of disease, disorder, or condition; delay orslowing the progress of the disease, disorder, or condition;amelioration or palliation of the disease, disorder, or condition; andremission (whether partial or total), whether detectable orundetectable. “Palliating” a disease, disorder, or condition means thatthe extent and/or undesirable clinical manifestations of the disease,disorder, or condition are lessened and/or time course of theprogression is slowed or lengthened, as compared to the extent or timecourse in the absence of treatment. By “treating cancer,” “preventingcancer,” or “inhibiting cancer” is meant causing a reduction in the sizeof a tumor or the number of cancer cells, slowing or inhibiting anincrease in the size of a tumor or cancer cell proliferation, increasingthe disease-free survival time between the disappearance of a tumor orother cancer and its reappearance, preventing or reducing the likelihoodof an initial or subsequent occurrence of a tumor or other cancer, orreducing an adverse symptom associated with a tumor or other cancer. Ina desired embodiment, the percent of tumor or cancerous cells survivingthe treatment is at least 20, 40, 60, 80, or 100% lower than the initialnumber of tumor or cancerous cells, as measured using any standardassay. Desirably, the decrease in the number of tumor or cancerous cellsinduced by administration of a compound of the invention is at least 2,5, 10, 20, or 50-fold greater than the decrease in the number ofnon-tumor or non-cancerous cells. Desirably, the methods of the presentinvention result in a decrease of 20, 40, 60, 80, or 100% in the size ofa tumor or number of cancerous cells, as determined using standardmethods. Desirably, at least 20, 40, 60, 80, 90, or 95% of the treatedsubjects have a complete remission in which all evidence of the tumor orcancer disappears. Desirably, the tumor or cancer does not reappear orreappears after no less than 5, 10, 15, or 20 years. By“prophylactically treating” a disease or condition (e.g., cancer) in asubject is meant reducing the risk of developing (i.e., the incidence)of or reducing the severity of the disease or condition prior to theappearance of disease symptoms. The prophylactic treatment maycompletely prevent or reduce appears of the disease or a symptom thereofand/or may be therapeutic in terms of a partial or complete cure for adisease and/or adverse effect attributable to the disease. Prophylactictreatment may include reducing or preventing a disease or condition(e.g., preventing cancer) from occurring in an individual who may bepredisposed to the disease but has not yet been diagnosed as having it.

Composition of Particles Comprising Anionic Agents

In some embodiments, a particle of the invention includes a cationiclipid (e.g., DODMA, DOTMA, DPePC, DODAP, or DOTAP), a neutral lipid(e.g., DSPC, POPC, DOPE, or SM), and, optionally, a sterol derivative(e.g., cholesterol; cholestanone; cholestenone; coprostanol;3β-[-(N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-cholesterol); bis-guanidium-tren-cholesterol (BGTC);(2S,3S)-2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)carbonylamino)ethyl2,3,4,4-tetrahydroxybutanoate (DPC-1);(2S,3S)-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)2,3,4,4-tetrahydroxybutanoate (DPC-2);bis((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)2,3,4-trihydroxypentanedioate (DPC-3); or6-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)oxidophosphoryloxy)-2,3,4,5-tetrahydroxyhexanoate(DPC-4)). In some embodiments, the particle further includes a PEG-lipidconjugate (e.g., PEG-DMG, PEG-DMPE, PEG-DPPE, PEG-DPG, PEG-DOPE, orPEG-DOG).

In some embodiments, the particle includes from about 10 mol % to about40 mol % of one or more compounds of the invention (e.g., one or more ofany compounds described herein, e.g., in Table 1), from about 10 mol %to about 40 mol % of one or more cationic lipids or one or morecompounds of the invention (e.g., one or more of any compounds describedherein, e.g., in Table 1), from about 1 mol % to about 20 mol % of oneor more PEG-lipid conjugates, from about 5 mol % to about 20 mol % ofone or more neutral lipids, and from about 20 mol % to about 40 mol % ofone or more sterol derivatives.

In particular embodiments, the particle includes from about 10 mol % toabout 80 mol % (e.g., from about 40 mol % to about 55 mol %, such asabout 48 mol %) of one or more cationic lipids (e.g., compounds of theinvention and/or other cationic lipids, as described herein), from about1 mol % to about 20 mol % of one or more PEG-lipid conjugates, fromabout 5 mol % to about 20 mol % of one or more neutral lipids, and fromabout 20 mol % to about 40 mol % of one or more sterol derivatives. Insome embodiments, the particle includes from about 10 mol % to about 30mol % (e.g., about 22 mol %) of one or more compounds of the invention(e.g., L-6, L-30, and/or any described herein), from about 15 mol % toabout 35 mol % (e.g., about 26 mol %) of one or more cationic lipids(e.g., DODMA or any described herein), from about 3 mol % to about 9 mol% (e.g., about 6 mol %) of one or more PEG-lipid conjugates (e.g.,PEG-DSPE, PEG-DMPE, and/or any described herein), from about 10 mol % toabout 20 mol % (e.g., about 14 mol %) of one or more neutral lipids(e.g., DSPC or any described herein), and from about 20 mol % to about40 mol % (e.g., from about 29 mol % to about 33 mol %, such as about 33mol %) of one or more sterol derivatives (e.g., cholesterol, aderivative thereof, or any described herein).

In some embodiments, one or more compounds of Table 1 is present in anamount between about 10 mol % to about 40 mol %, e.g., between about 10mol % and about 15 mol %, between about 10 mol % and about 20 mol %,between about 10 mol % and about 25 mol %, between about 10 mol % andabout 30 mol %, between about 10 mol % and about 35 mol %, between about15 mol % and about 20 mol %, between about 15 mol % and about 25 mol %,between about 15 mol % and about 30 mol %, between about 15 mol % andabout 35 mol %, between about 15 mol % and about 40 mol %, between about20 mol % and about 25 mol %, between about 20 mol % and about 30 mol %,between about 20 mol % and about 35 mol %, between about 20 mol % andabout 40 mol %, between about 25 mol % and about 30 mol %, between about25 mol % and about 35 mol %, between about 25 mol % and about 40 mol %,between about 30 mol % and about 35 mol %, between about 30 mol % andabout 40 mol %, or between about 35 mol % and about 40 mol % (e.g.,about 21.0 mol %, 21.2 mol %, 21.4 mol %, 21.6 mol %, 21.8 mol %, 22 mol%, 25 mol %, 26 mol %, 26 mol %, 30 mol %, 35 mol %, or 40 mol %) of oneor more compounds of Table 1. In some embodiments, one or more compoundsof Table 1 is present in an amount between about 10 mol % to about 80mol %, e.g., between about 10 mol % and about 15 mol %, between about 10mol % and about 20 mol %, between about 10 mol % and about 25 mol %,between about 10 mol % and about 30 mol %, between about 10 mol % andabout 35 mol %, between about 10 mol % and about 40 mol %, between about10 mol % and about 45 mol %, between about 10 mol % and about 50 mol %,between about 10 mol % and about 55 mol %, between about 10 mol % andabout 60 mol %, between about 10 mol % and about 65 mol %, between about10 mol % and about 70 mol %, between about 10 mol % and about 75 mol %,between about 15 mol % and about 20 mol %, between about 15 mol % andabout 25 mol %, between about 15 mol % and about 30 mol %, between about15 mol % and about 35 mol %, between about 15 mol % and about 40 mol %,between about 15 mol % and about 45 mol %, between about 15 mol % andabout 50 mol %, between about 15 mol % and about 55 mol %, between about15 mol % and about 60 mol %, between about 15 mol % and about 65 mol %,between about 15 mol % and about 70 mol %, between about 15 mol % andabout 75 mol %, between about 15 mol % and about 80 mol %, between about20 mol % and about 25 mol %, between about 20 mol % and about 30 mol %,between about 20 mol % and about 35 mol %, between about 20 mol % andabout 40 mol %, between about 20 mol % and about 45 mol %, between about20 mol % and about 50 mol %, between about 20 mol % and about 55 mol %,between about 20 mol % and about 60 mol %, between about 20 mol % andabout 65 mol %, between about 20 mol % and about 70 mol %, between about20 mol % and about 75 mol %, between about 20 mol % and about 80 mol %,between about 25 mol % and about 30 mol %, between about 25 mol % andabout 35 mol %, between about 25 mol % and about 40 mol %, between about25 mol % and about 45 mol %, between about 25 mol % and about 50 mol %,between about 25 mol % and about 55 mol %, between about 25 mol % andabout 60 mol %, between about 25 mol % and about 65 mol %, between about25 mol % and about 70 mol %, between about 25 mol % and about 75 mol %,between about 25 mol % and about 80 mol %, between about 30 mol % andabout 35 mol %, between about 30 mol % and about 40 mol %, between about30 mol % and about 45 mol %, between about 30 mol % and about 50 mol %,between about 30 mol % and about 55 mol %, between about 30 mol % andabout 60 mol %, between about 30 mol % and about 65 mol %, between about30 mol % and about 70 mol %, between about 30 mol % and about 75 mol %,between about 30 mol % and about 80 mol %, between about 35 mol % andabout 40 mol %, between about 35 mol % and about 45 mol %, between about35 mol % and about 50 mol %, between about 35 mol % and about 55 mol %,between about 35 mol % and about 60 mol %, between about 35 mol % andabout 65 mol %, between about 35 mol % and about 70 mol %, between about35 mol % and about 75 mol %, or between about 35 mol % and about 80 mol%, between about 40 mol % and about 45 mol %, between about 40 mol % andabout 50 mol %, between about 40 mol % and about 55 mol %, between about40 mol % and about 60 mol %, between about 40 mol % and about 65 mol %,between about 40 mol % and about 70 mol %, between about 40 mol % andabout 75 mol %, between about 40 mol % and about 80 mol %, between about45 mol % and about 50 mol %, between about 45 mol % and about 55 mol %,between about 45 mol % and about 60 mol %, between about 45 mol % andabout 65 mol %, between about 45 mol % and about 70 mol %, between about45 mol % and about 75 mol %, or between about 45 mol % and about 80 mol%, between about 50 mol % and about 55 mol %, between about 50 mol % andabout 60 mol %, between about 50 mol % and about 65 mol %, between about50 mol % and about 70 mol %, between about 50 mol % and about 75 mol %,or between about 50 mol % and about 80 mol % (e.g., about 21.0 mol %,21.2 mol %, 21.4 mol %, 21.6 mol %, 21.8 mol %, 22 mol %, 25 mol %, 26mol %, 26 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 48 mol %, 49mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, or 75 mol %) ofone or more compounds of Table 1.

In some embodiments, one or more cationic lipids is present in an amountbetween about 10 mol % to about 40 mol %, e.g., between about 10 mol %and about 15 mol %, between about 10 mol % and about 20 mol %, betweenabout 10 mol % and about 25 mol %, between about 10 mol % and about 30mol %, between about 10 mol % and about 35 mol %, between about 15 mol %and about 20 mol %, between about 15 mol % and about 25 mol %, betweenabout 15 mol % and about 30 mol %, between about 15 mol % and about 35mol %, between about 15 mol % and about 40 mol %, between about 20 mol %and about 25 mol %, between about 20 mol % and about 30 mol %, betweenabout 20 mol % and about 35 mol %, between about 20 mol % and about 40mol %, between about 25 mol % and about 30 mol %, between about 25 mol %and about 35 mol %, between about 25 mol % and about 40 mol %, betweenabout 30 mol % and about 35 mol %, between about 30 mol % and about 40mol %, or between about 35 mol % and about 40 mol % (e.g., about 25.1mol %, 25.2 mol %, 25.3 mol %, 25.4 mol %, 25.5 mol %, 25.6 mol %, 25.7mol %, 25.8 mol %, 25.9 mol %, 26.0 mol %, 26.2 mol %, 26.4 mol %, 26.6mol %, 26.8 mol %, or 27 mol %) of one or more cationic lipids (e.g.,DODMA or any described herein, such as in Table 1).

In some embodiments, one or more PEG-lipid conjugates is present in anamount between about 1 mol % to about 20 mol %, e.g., between about 1mol % and about 5 mol %, between about 1 mol % and about 10 mol %,between about 1 mol % and about 15 mol %, between about 2 mol % andabout 5 mol %, between about 2 mol % and about 10 mol %, between about 2mol % and about 15 mol %, between about 2 mol % and about 20 mol %,between about 5 mol % and about 10 mol %, between about 5 mol % andabout 15 mol %, between about 5 mol % and about 20 mol %, between about10 mol % and about 15 mol %, between about 10 mol % and about 20 mol %,between about 15 mol % and about 20 mol % (e.g., about 2.5 mol %, 2.6mol %, 2.7 mol %, 2.8 mol %, 2.9 mol %, 3 mol %, 3.5 mol %, 4 mol %, 4.3mol %, 4.5 mol %, 4.7 mol %, 5 mol %, 5.3 mol %, 5.5 mol %, 5.7 mol %, 6mol %, 6.5 mol %, 6.7 mol %, 7 mol %, 7.5 mol %, 8 mol %, 8.5 mol %, or9 mol %) of one or more PEG-lipid conjugates (e.g., PEG-DSPE, PEG-DMPE,and/or any described herein).

In some embodiments, one or more neutral lipids is present in an amountbetween about 5 mol % to about 20 mol %, e.g., between about 5 mol % andabout 10 mol %, between about 5 mol % and about 15 mol %, between about5 mol % and about 20 mol %, between about 7 mol % and about 10 mol %,between about 7 mol % and about 15 mol %, between about 7 mol % andabout 20 mol %, between about 10 mol % and about 15 mol %, between about10 mol % and about 20 mol %, between about 15 mol % and about 20 mol %(e.g., about 13.0 mol %, 13.2 mol %, 13.4 mol %, 13.6 mol %, 13.8 mol %,14 mol %, 14.1 mol %, 14.3 mol %, 14.5 mol %, 14.7 mol %, or 14.9 mol %)of one or more neutral lipids (e.g., DSPC or any described herein).

In some embodiments, one or more sterol derivatives is present in anamount between about 20 mol % to about 40 mol %, e.g., between about 20mol % and about 25 mol %, between about 20 mol % and about 30 mol %,between about 20 mol % and about 35 mol %, between about 25 mol % andabout 30 mol %, between about 25 mol % and about 35 mol %, between about25 mol % and about 40 mol %, between about 30 mol % and about 35 mol %,between about 30 mol % and about 40 mol %, or between about 35 mol % andabout 40 mol % (e.g., about 28.4 mol %, 28.6 mol %, 28.8 mol %, 29.0 mol%, 30 mol %, 31 mol %, 32 mol %, 33 mol %, 33.2 mol %, 33.4 mol %, 33.6mol %, 33.8 mol %, 34 mol %, 34.4 mol %, 34.7 mol %, or 34.9 mol %) ofone or more sterol derivatives (e.g., cholesterol or any describedherein).

In some embodiments, the particle includes one or more lipid particlescomprising one or more RNA-binding agents and one or more transfectionlipids, where the one or more RNA-binding agents include from about 10mol % to about 40 mol % of one or more cationic lipids or one or morecompounds of Table 1 and from about 0.5 mol % to about 10 mol % of oneor more PEG-lipid; and where the one or more transfection lipids includefrom about 10 mol % to about 40 mol % of one or more compounds of Table1, from about 5 mol % to about 20 mol % of one or more neutral lipids,from about 0.5 mol % to about 10 mol % of one or more PEG-lipidconjugates, and from about 20 mol % to about 40 mol % of one or moresterol derivatives. Additional particles and percentages are asdescribed herein.

In some embodiments, the particle further includes an anionic agent,e.g., a polyanionic agent such as an RNAi agent (e.g., dsRNA, siRNA,miRNA, shRNA, ptgsRNA, or DsiRNA, e.g., DsiRNA) or an antisense agent.In some embodiments, the RNAi agent has a length of 10 to 40nucleotides, e.g., length of 10 to 15 nucleotides, 10 to 20 nucleotides,10 to 25 nucleotides, 10 to 30 nucleotides, 10 to 35 nucleotides, 15 to20 nucleotides, 15 to 25 nucleotides, 15 to 30 nucleotides, 15 to 35nucleotides, 15 to 40 nucleotides, 16 to 20 nucleotides, 16 to 25nucleotides, 16 to 30 nucleotides, 16 to 35 nucleotides, 16 to 40nucleotides, 20 to 25 nucleotides, 18 to 20 nucleotides, 18 to 25nucleotides, 18 to 30 nucleotides, 18 to 35 nucleotides, 18 to 40nucleotides, 19 to 20 nucleotides, 19 to 25 nucleotides, 19 to 30nucleotides, 19 to 35 nucleotides, 19 to 40 nucleotides, 20 to 30nucleotides, 20 to 35 nucleotides, 20 to 40 nucleotides, 25 to 30nucleotides, 25 to 35 nucleotides, 25 to 40 nucleotides, 30 to 35nucleotides, 30 to 40 nucleotides, or 35 to 40 nucleotides, e.g., alength of 25 to 35 nucleotides, e.g., a length of 16 to 30 nucleotides,e.g., a length of 19 to 29 nucleotides. In some embodiments, theantisense agent has a length of 8 to 50 nucleotides (e.g., a length of 8to 10 nucleotides, 8 to 15 nucleotides, 8 to 15 nucleotides, 8 to 20nucleotides, 8 to 25 nucleotides, 8 to 30 nucleotides, 8 to 35nucleotides, 8 to 40 nucleotides, or 8 to 45 nucleotides), e.g., alength of 14 to 35 nucleotides (e.g., a length of 14 to 15 nucleotides,14 to 20 nucleotides, 14 to 25 nucleotides, or 14 to 30 nucleotides),e.g., a length of 17 to 24 nucleotides, e.g., a length of 17 to 20nucleotides.

In some embodiments, the particle includes from about 1:10 (w/w) toabout 1:100 (w/w) ratio of the anionic agent to the total lipid presentin the particle, e.g., from about 1:10 (w/w) to about 1:15 (w/w) ratio,from about 1:10 (w/w) to about 1:20 (w/w) ratio, from about 1:10 (w/w)to about 1:40 (w/w) ratio, from about 1:10 (w/w) to about 1:50 (w/w)ratio, from about 1:10 (w/w) to about 1:60 (w/w) ratio, from about 1:10(w/w) to about 1:70 (w/w) ratio, from about 1:10 (w/w) to about 1:80(w/w) ratio, from about 1:10 (w/w) to about 1:90 (w/w) ratio, from about1:10 (w/w) to about 1:95 (w/w) ratio, from about 1:20 (w/w) to about1:40 (w/w) ratio, from about 1:20 (w/w) to about 1:50 (w/w) ratio, fromabout 1:20 (w/w) to about 1:60 (w/w) ratio, from about 1:20 (w/w) toabout 1:70 (w/w) ratio, from about 1:20 (w/w) to about 1:80 (w/w) ratio,from about 1:20 (w/w) to about 1:90 (w/w) ratio, from about 1:20 (w/w)to about 1:95 (w/w) ratio, from about 1:20 (w/w) to about 1:100 (w/w)ratio, from about 1:40 (w/w) to about 1:50 (w/w) ratio, from about 1:40(w/w) to about 1:60 (w/w) ratio, from about 1:40 (w/w) to about 1:70(w/w) ratio, from about 1:40 (w/w) to about 1:80 (w/w) ratio, from about1:40 (w/w) to about 1:90 (w/w) ratio, from about 1:40 (w/w) to about1:95 (w/w) ratio, from about 1:40 (w/w) to about 1:100 (w/w) ratio, fromabout 1:50 (w/w) to about 1:60 (w/w) ratio, from about 1:50 (w/w) toabout 1:70 (w/w) ratio, from about 1:50 (w/w) to about 1:80 (w/w) ratio,from about 1:50 (w/w) to about 1:90 (w/w) ratio, from about 1:50 (w/w)to about 1:95 (w/w) ratio, from about 1:50 (w/w) to about 1:100 (w/w)ratio, from about 1:60 (w/w) to about 1:70 (w/w) ratio, from about 1:60(w/w) to about 1:80 (w/w) ratio, from about 1:60 (w/w) to about 1:90(w/w) ratio, from about 1:60 (w/w) to about 1:95 (w/w) ratio, from about1:60 (w/w) to about 1:100 (w/w) ratio, from about 1:80 (w/w) to about1:90 (w/w) ratio, from about 1:80 (w/w) to about 1:95 (w/w) ratio, orfrom about 1:80 (w/w) to about 1:100 (w/w) ratio of the anionic agent tothe total lipid present in the particle.

In some embodiments, the particle includes a liposome (e.g., a lipidnanoparticle), a lipoplex, or a micelle.

In one aspect, the process of the invention features a pharmaceuticalcomposition including any compound described herein (e.g., one or morecompound provided in Table 1), or a pharmaceutically acceptable saltthereof, or any particle or formulation described herein; and apharmaceutically acceptable excipient.

In another aspect, the invention features a method of treating orprophylactically treating a disease in a subject, the method includingadministering to the subject a particle made by a process describedherein (e.g., as set forth in the below Examples), any formulationdescribed herein, or any composition described in an amount sufficientto treat the disease (e.g., liver cancer (e.g., hepatocellularcarcinoma, hepatoblastoma, cholangiocarcinoma, angiosarcoma, orhemangiosarcoma), lung cancer (e.g., small cell lung cancer, non smallcell lung cancer), prostate cancer, or neuroblastoma). The inventionfurther features a method of treating or prophylactically treatingneoplastic diseases and associated complications including, but notlimited to, carcinomas (e.g., lung, breast, pancreatic, colon,hepatocellular, renal, female genital tract, prostate, squamous cell,carcinoma in situ), lymphoma (e.g., histiocytic lymphoma, non-Hodgkin'slymphoma), MEN2 syndromes, neurofibromatosis (including Schwann cellneoplasia), myelodysplastic syndrome, leukemia, tumor angiogenesis,cancers of the thyroid, liver, bone, skin, brain, central nervoussystem, pancreas, lung (e.g., small cell lung cancer, non small celllung cancer), breast, colon, bladder, prostate, gastrointestinal tract,endometrium, fallopian tube, testes and ovary, gastrointestinal stromaltumors (GISTs), prostate tumors, mast cell tumors (including canine mastcell tumors), acute myeloid myelofibrosis, leukemia, acute lymphocyticleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia,multiple myeloma, melanoma, mastocytosis, gliomas, glioblastoma,astrocytoma, neuroblastoma, sarcomas (e.g., sarcomas of neuroectodermalorigin or leiomyosarcoma), metastasis of tumors to other tissues, andchemotherapy-induced hypoxia.

In another aspect, the invention features a method of modulating theexpression of a target nucleic acid in a subject, the method includingadministering any particle made by a process described herein (e.g., asset forth in the below Examples), any formulation described herein, orany composition described in an amount sufficient to reduce theexpression of the target gene (e.g., any described herein, e.g., one ormore target genes selected from the group consisting of ABL1, AR,β-Catenin (CTNNB1), BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA1, ERBA2,ERBB1, ERBB2, ERBB3, ERBB4, ETS1, ETS2, ETV6, FGR, FOS, FYN, HCR, HRAS,JUN, KRAS, LCK, LYN, MET, MDM2, MLL1, MLL2, MLL3, MYB, MYC, MYCL1, MYCN,NRAS, PIM1, PML, RET, SRC, TAL1, TAL2, TCL3, TCL5, YES, BRCA1, BRCA2,MADH4, MCC, NF1, NF2, RB1, TP53, WT1, ApoB100, CSN5, CDK6, ITGB1, TGFβ1,Cyclin D1, hepcidin, PCSK9, TTR, PLK1, and KIF1-binding protein) in thesubject (e.g., where the method includes reducing the expression of thetarget gene in the subject).

In another embodiment, the invention features the administration of adosage of the particle/anionic agent of the invention to a subject oneor more times per day (e.g., 1, 2, 3, or 4 times per day), one or moretimes per week (e.g., 2, 3, 4, 5, 6, or 7 times per week) or one or moretimes per month (e.g., 2, 3, 4, 5, 6, 7, or 10 times per month). Asubject may receive dosages of the anionic agent in the range of about0.0001 to about 10 mg/kg, e.g., about 0.0001 to about 1 mg/kg, about0.0001 to about 5 mg/kg, about 0.001 to about 1 mg/kg, about 0.001 toabout 5 mg/kg, about 0.001 to about 10 mg/kg, about 0.01 to about 1mg/kg, about 0.01 to about 5 mg/kg, about 0.01 to about 10 mg/kg, about1 to about 5 mg/kg, or about 1 to about 10 mg/kg, in any dosage regimen(e.g., one or more times per day (e.g., 1, 2, 3, or 4 times per day),one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 times per week) orone or more times per month (e.g., 2, 3, 4, 5, 6, 7, or 10 times permonth)).

In certain embodiments, a subject may receive dosages of a particle madeby a process of the invention in the range of about 0.001 to about 200mg/kg, e.g., about 0.001 to about 1 mg/kg, about 0.001 to about 10mg/kg, about 0.001 to about 20 mg/kg, about 0.001 to about 50 mg/kg,about 0.001 to about 100 mg/kg, about 0.01 to about 1 mg/kg, about 0.01to about 10 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 50mg/kg, about 0.01 to about 100 mg/kg, about 0.01 to about 200 mg/kg,about 0.1 to about 1 mg/kg, about 0.1 to about 10 mg/kg, about 0.1 toabout 20 mg/kg, about 0.1 to about 50 mg/kg, about 0.1 to about 100mg/kg, about 0.1 to about 200 mg/kg, about 1 to about 10 mg/kg, about 1to about 20 mg/kg, about 1 to about 50 mg/kg, about 1 to about 100mg/kg, about 1 to about 200 mg/kg, about 10 to about 20 mg/kg, about 10to about 50 mg/kg, about 10 to about 100 mg/kg, about 10 to about 200mg/kg, about 20 to about 50 mg/kg, about 20 to about 100 mg/kg, or about20 to about 200 mg/kg, in any dosage regimen (e.g., one or more timesper day (e.g., 1, 2, 3, or 4 times per day), one or more times per week(e.g., 2, 3, 4, 5, 6, or 7 times per week) or one or more times permonth (e.g., 2, 3, 4, 5, 6, 7, or 10 times per month)).

In another aspect, the invention features a method of delivering aparticle/agent made by a process of the invention to a specific type oftissue. Examples of specific types of tissues to which theparticle/agent may be delivered to include, but are not limited to,liver, pancreas, lung, prostate, kidney, bone marrow, spleen, thymus,lymph node, brain, spinal cord, heart, skeletal muscle, skin, oralmucosa, esophagus, stomach, ileum, small intestine, colon, bladder,cervix, ovary, testis, mammary gland, adrenal gland, adipose tissue(white and/or brown), blood (e.g., hematopoietic cells, such as humanhematopoietic progenitor cells, human hematopoietic stem cells, CD34+cells, CD4+ cells), lymphocytes, and other blood lineage cells.

Amino-Amine and Amino-Amide Lipids

Exemplary compounds employed in the processes of the invention are shownin Table 1.

TABLE 1 L-1

L-2

L-3

L-4

L-5

L-6

L-7

L-8

L-9

L-10

L-11

L-12

L-13

L-14

L-15

L-16

L-17

L-18

L-19

L-20

L-21

L-22

L-23

L-24

L-25

L-26

L-27

L-28

L-29

L-30

L-31

L-32

L-33

L-34

L-35

L-36

L-37

L-38

L-39

L-40

L-41

L-42

L-43

L-44

L-45

L-46

L-47

L-48

L-49

Certain compounds of the processes of the invention (e.g., as providedin Table 1) may be prepared by processes analogous to those establishedin the art, for example, by the reaction sequences shown in Schemes 1-4.

The secondary amine of formula C1 may be prepared under reductiveamination conditions by treating ketone A1, where R¹ and R² is a lipidtail group, as described herein, with a primary amine B1, wherein R⁴ isdescribed herein. Conditions for reductive amination include combiningketone A1 and primary amine B1 with a reducing agent, such as sodiumcyanoborohydride or sodium trioacetoxyborohydride, in an appropriatesolvent. In particular embodiments, the amino-amine lipid of C1 isfurther oxidized to form a corresponding amino-amide lipid having an oxogroup on the carbon in R³ that is adjacent to the nitrogen. In otherembodiments, the amino-amine lipid of C1 is further subject toalkylation at the nitrogen or on any carbon in R⁴. Exemplary compoundsthat can be produced using this scheme are provided in Tables 1-4.

The tertiary amine of formula E2 may be prepared under reductiveamination conditions by treating ketone A2, where each R¹ and R² is alipid tail group, as described herein, with a secondary amine D2, whereR³ and R⁴ is described herein. Conditions for reductive aminationinclude combining ketone A2 and secondary amine D2 with a reducingagent, such as sodium cyanoborohydride or sodium trioacetoxyborohydride,in an appropriate solvent. In some embodiments of D2, R³ and R⁴ join toform a heterocyclic ring containing one or more heteroatoms, and theresultant tertiary amine E2 includes such R³ and R⁴ groups. Inparticular embodiments, the amino-amine lipid of E2 is further oxidizedto form a corresponding amino-amide lipid having an oxo group on acarbon in R³ or R⁴ that is adjacent to the nitrogen. In otherembodiments, the amino-amine lipid of E2 is further subject toalkylation on any carbon in R³ and/or R⁴.

The amine of formula F3 may be prepared by combining ketone A3, ammonia,dihydrogen, and a catalyst in an appropriate solvent, optionally, underhigh pressure. The amino-amide lipid of formula H3 may be prepared bycombining amine F3 with an activated carboxylic acid G3 in anappropriate solvent, where LG is a leaving group and R⁴ is describedherein. Exemplary LG's include halo (e.g., chloride, bromine, oriodine), tosylate, and triflate. The amino-amine lipid of I3 may beprepared by combining amide H3 with a reducing agent (e.g., lithiumaluminum hydride, borane-tetrahydrofuran, or borane-dimethylsulfide). Inparticular embodiments, the amino-amide lipid of H3 is further subjectto alkylation at the nitrogen or on any carbon in R^(4′). In otherembodiments, the amino-amine lipid of I3 is further subject toalkylation at the nitrogen or on any carbon in R⁴.

The amino-amide lipid of formula K4 may be prepared by combining ketoneA4 and amine J4 in an appropriate solvent, where LG is a leaving groupand R¹, R², and R⁴ are described herein. Exemplary LG's include halo(e.g., chloride, bromine, or iodine), tosylate, and triflate. Theamino-amine lipid of L4 may be prepared by combining amide K4 with areducing agent (e.g., lithium aluminum hydride, borane-tetrahydrofuran,or borane-dimethylsulfide). In other embodiments, the amino-amide lipidof K4 is further subject to alkylation at the nitrogen or on any carbonin R^(4′). In other embodiments, the amino-amine lipid of L4 is furthersubject to alkylation at the nitrogen or on any carbon in R⁴.

In any of the above schemes, R⁴ can be optionally substitutedheterocyclyl, optionally substituted -L′-NR⁵R6⁵, optionally substituted—C(O)-L¹-NR⁵R⁶, or optionally substituted -L¹-heterocyclyl, as describedherein.

In any of the above schemes, the compounds can be further alkylated tointroduce an optionally substituted C₁₋₆ alkyl on N (i.e., R³ is anoptionally substituted C₁₋₆ alkyl) to form a tertiary amine.

Any of the lipids described herein, e.g., as in Table 1, can be producedby applying the synthetic schemes provided above, synthetic schemesdisclosed in the art and, if needed, by making modifications known toone skilled in the art.

Lipid Head Groups

Compounds employed in the processes of the invention may include a lipidhead group, a headpiece, and one or more lipid tail groups. Theheadpiece, e.g., >CH—, connects the head group to the tail group(s). Inparticular embodiments, the head group includes two or more nitrogenatoms. Any of the head groups described herein, e.g., in Tables 2 or 3,may be optionally substituted with one or more substituents (e.g., oneor more substituents described herein for alkyl).

A non-limiting list of head groups having an amine group is provided inTable 2. Any of the head groups described herein, e.g., head groups H-1to H-39 in Table 2, can be combined with any of the tail groupsdescribed herein, e.g., in Table 4, via headpiece >CH— to form acompound of the invention.

TABLE 2 Examples of lipid head groups

(H-1)

(H-2)

(H-3)

(H-4)

(H-5)

(H-6)

(H-7)

(H-8)

(H-9)

(H-10)

(H-11)

(H-12)

(H-13)

(H-14)

(H-15)

(H-16)

(H-17)

(H-18)

(H-19)

(H-20)

(H-21)

(H-22)

(H-23)

(H-24)

(H-25)

(H-26)

(H-27)

(H-28)

(H-29)

(H-30)

(H-31)

(H-32)

(H-33)

(H-34)

(H-35)

(H-36)

(H-37)

(H-38)

(H-39)

A non-limiting list of head groups having an amide group is provided inTable 3. Any of the head groups described herein, e.g., head groups H-40to H-52 in Table 3, can be combined with any of the tail groupsdescribed herein, e.g., in Table 4, via headpiece >CH— to form acompound for use in the processes of the invention.

TABLE 3 Examples of lipid head groups containing an amide

(H-40)

(H-41)

(H-42)

(H-43)

(H-44)

(H-45)

(H-46)

(H-47)

(H-48)

(H-49)

(H-50)

(H-51)

(H-52)

Lipid Tail Groups

As described herein, the compounds used in the processes of theinvention generally include one or more tail groups that can optionallyinclude one or more heteroatoms. For each compound, the tail groups canbe the same or different. Any of the tail groups described herein, e.g.,in Table 4, may be optionally substituted with one or more substituents(e.g., one or more substituents described herein for alkyl).

Exemplary tail groups include saturated and unsaturated groups havingcarbon or one or more heteroatoms (e.g., O), such as linolenyl (C18:3),linolenyloxy (C18:3), linolenoyl (C18:3), linoleyl (C18:2), linoleyloxy(C18:2), and linoleoyl (C18:2); and any heteroatomic tail groupdescribed herein that is connected to the headpiece by a methylene,e.g., tail groups selected from the group of linolenyloxymethylene(C18:3), linolenoylmethylene (C18:3), and linoleyloxymethylene (C18:2),or linoleoylmethylene (C18:2). Additional non-limiting list of lipidtail groups is provided in Table 4.

TABLE 4 Examples of lipid tail groups linolenyl (C18:3)

linolenyloxy (C18:3)

linolenoyl (C18:3)

linoleyl (C18:2)

linoleyloxy (C18:2)

linoleoyl C18:2)

oleyl (C18:1)

oleyloxy (18:1)

oleyloxymethylene (18:1)

oleoyl (C18:1)

oleoylmethylene (C18:1)

stearyl (18:0)

stearyloxy (C18:0)

stearoyl (C18:0)

palmityl (16:0)

palmityloxy (C16:0)

palmitoyl (C16:0)

palmitoylmethylene (C16:0)

myristyl (14:0)

myristyloxy (C14:0)

myristoyl (C14:0)

lauryl (12:0)

lauryloxy (12:0)

lauryloyl (12:0)

Measurement of pKa Values of Lipids in Assembled Nanoparticles

Different physiochemical properties of lipids greatly determine thebehavior of lipids when present in different environments. One suchimportant property is the ionization constant (Ka) of the lipid. Theintrinsic pKa of the lipid may not be a correct representation of theirbehavior when present in an assembled nanoparticle. When present in anaqueous environment, the lipid experiences an environment with highdielectric constant, whereas in an assembled nanoparticle/vesicle, it issurrounded by lipids which provide low dielectric constant. In addition,the surrounding lipids, cholesterol, and PEGylated lipids all influencethe apparent pKa of the formulation. The nature of interaction betweenthe cationic lipids and nucleic acid being electrostatic, the apparentpKa of the formulation determines encapsulation of nucleic acid in thenanoparticle and also its subsequent intracellular release.

The TNS fluorescence method may be used to determine the apparent pKa ofthe lipid in the formulation. TNS (2-(p-toluidino)-6-naphthalenesulfonic acid) is a negatively charged fluorescent dye whosefluorescence is quenched in the presence of water. TNS partitions into apositively charged membrane and this results in an increase influorescence due to removal of water. The increase in the fluorescencecan thus be used to estimate the ionization of a cationic lipid whenpresent in different pH environment. Methods of determining pKa usingTNS are known in the art.

Methods of Determining Solubility

The compounds used in the processes of the invention, as well asparticles and compounds resulting from the processes of the invention,can be assessed to determine its solubility in a particular solvent.Compounds of the invention include but are not limited to any lipidmolecule (e.g., cationic, anionic, or neutral lipid), sterol, component,particle, or combination thereof, as described herein.

Solubility can be measured by any useful method and/or by any usefulmetric. Exemplary methods and metrics include high performance liquidchromatography (optionally coupled with an evaporative light scatteringdetector), nuclear magnetic resonance, mass spectrometry, UV/VISspectroscopy, and metrics such as the partition coefficient (Log P),solubility (e.g., as measured by g of solute per kg of solvent, g ofsolute per dL (100 mL) of solvent, molarity, molality, or molefraction), critical micelle concentration, average particle size, sizedistribution of particles (e.g., as determined by the polydispersityindex), homogeneity of the resultant solution, and encapsulationefficiency (e.g., of an anionic agent, such as any described herein,e.g., DsiRNA).

Formulations

The compounds used in the processes of the invention to synthesizeparticles and/or the particles may be combined with one or more lipidmolecules (e.g., cationic, anionic, or neutral lipids) to produce aformulation, or the particles may be the formulation. The formulationcan also include one or more components (e.g., sterol derivatives,PEG-lipid conjugates, polyamide-lipid conjugates, gangliosides,antioxidants, surfactants, amphiphilic agents, or salts) and/or one ormore anionic agents (e.g., one or more nucleic acids or RNAi agents).Methods of formulating lipids to incorporate nucleic acid agents havebeen described, see, for example, Judge et al., J. Clin. Invest.119(3):661, 2009; Noble et al., Cancer Chemother. Pharmacol. 64(4):741,2009; Abrams et al., Mol. Ther. 18(1):171, 2009; Yagi et al., CancerRes. 69(16):6531, 2009; Ko et al., J. Control. Release 133(2):132, 2009;Mangala et al., Methods Mol. Biol. 555:29, 2009, which are herebyincorporated by reference.

Formulations with More than One Lipid Molecule

Formulations incorporating the particles of the processes of theinvention may include any useful combination of lipid molecules (e.g., acompound as tabulated herein, a cationic lipid (optionally including oneor more cationic lipids, e.g., one or more cationic lipids as describedherein and/or optionally including one or more cationic lipids known inthe art), a neutral lipid, an anionic lipid, and a PEG-lipid conjugate),including polypeptide-lipid conjugates and other components that aid inthe formation or stability of a lipid vector, as described herein. Theformulations incorporating the particles of the processes of theinvention may include other components that aid in formation orstability.

The percentage of each component in the formulation can be balanced toproduce a particle or lipid vector capable of encapsulating an anionicagent and transfecting the agent into a cell. An exemplary formulationincludes from about 10 mol % to about 40 mol % of one or more compoundsof Table 1, from about 10 mol % to about 40 mol % of one or morecationic lipids, from about 1 mol % to about 20 mol % of one or morePEG-lipid conjugates, from about 5 mol % to about 20 mol % of one ormore neutral lipids, and from about 20 mol % to about 40 mol % of one ormore sterol derivatives. In particular embodiments, the formulationincludes from about 20 mol % to about 25 mol % (e.g., about 21.0 mol %,21.2 mol %, 21.4 mol %, 21.6 mol %, 21.8 mol %, or 22 mol %) of one ormore compounds of Table 1, from about 25 mol % to about 30 mol % (e.g.,about 25.1 mol %, 25.2 mol %, 25.3 mol %, 25.4 mol %, 25.5 mol %, 25.6mol %, 25.7 mol %, 25.8 mol %, 25.9 mol %, 26.0 mol %, 26.2 mol %, 26.4mol %, 26.6 mol %, 26.8 mol %, or 27 mol %) of one or more cationiclipids (e.g., DODMA), from about 10 mol % to about 15 mol % (e.g., about13.0 mol %, 13.2 mol %, 13.4 mol %, 13.6 mol %, 13.8 mol %, 14 mol %,14.1 mol %, 14.3 mol %, 14.5 mol %, 14.7 mol %, or 14.9 mol %) of one ormore neutral lipids (e.g., DSPC), from about 2.5 mol % to about 10 mol %(e.g., about 2.5 mol %, 2.6 mol %, 2.7 mol %, 2.8 mol %, 2.9 mol %, 3mol %, 3.5 mol %, 4 mol %, 4.3 mol %, 4.5 mol %, 4.7 mol %, 5 mol %, 5.3mol %, 5.5 mol %, 5.7 mol %, 6 mol %, 6.5 mol %, 6.7 mol %, 7 mol %, 7.5mol %, 8 mol %, 8.5 mol %, or 9 mol %) of one or more PEG-lipidconjugates (e.g., about 2.8 mol %, 2.9 mol %, 3.0 mol %, 3.5 mol %, 3.7mol %, 3.9 mol %, 4 mol %, 4.1 mol %, 4.3 mol %, 4.5 mol %, 4.7 mol %,4.9 mol %, 5 mol %, 5.1 mol %, 5.3 mol %, 5.5 mol %, 5.7 mol %, 5.9 mol%, 6 mol %, 6.3 mol %, 6.5 mol %, 6.7 mol %, or 7 mol % of PEG2000-DSPEand/or PEG2000-DMPE and/or 3 mol %, 3.5 mol %, 3.7 mol %, 3.9 mol %, 4mol %, 4.1 mol %, 4.3 mol %, 4.5 mol %, 4.7 mol %, 4.9 mol %, 5 mol %,5.1 mol %, 5.3 mol %, 5.5 mol %, 5.7 mol %, 5.9 mol %, 6 mol %, 6.3 mol%, 6.5 mol %, 6.7 mol %, or 7 mol % of PEG2000-DMG), and about 25 mol %to about 35 mol % (e.g., about 28.4 mol %, 28.6 mol %, 28.8 mol %, 29.0mol %, 30 mol %, 31 mol %, 32 mol %, 33 mol %, 33.2 mol %, 33.4 mol %,33.6 mol %, 33.8 mol %, 34 mol %, 34.4 mol %, 34.7 mol %, or 34.9 mol %)of a sterol derivative (e.g., cholesterol).

The formulation can include any useful amount of one or more cationiclipids. In some embodiments, the content of the cationic lipid in theformulation is from about 10 mol % to about 40 mol % (e.g., from about10 mol % to 15 mol %, from about 15 mol % to 20 mol %, from about 20 mol% to 25 mol %, from about 25 mol % to 30 mol %, from about 30 mol % to35 mol %, and from about 35 mol % to 40 mol %). In particularembodiments, mixed cationic lipids (e.g., 10.8 mol % of L-1 and 10.8 mol% of L-2) are used.

In some embodiments, the formulation includes lipid particles having oneor more RNA-binding agents and one or more transfection lipids, wherethe one or more RNA-binding agents include about 10 mol % to about 40mol % of one or more cationic lipids (e.g., DODMA) and about 0.5 mol %to about 10 mol % of one or more PEG-lipid conjugates (e.g., PEG-DSPE,such as PEG2000-DSPE, and/or PEG-DMPE, such as PEG2000-DMPE); and wherethe one or more transfection lipids include about 10 mol % to about 40mol % of one or more compounds of Table 1 (e.g., L-6, -30, or any inTable 1), about 5 mol % to about 20 mol % of one or more neutral lipids(e.g., DSPC), about 0.5 mol % to about 10 mol % of one or more PEG-lipidconjugates (e.g., PEG-DSPE, such as PEG2000-DSPE, and/or PEG-DMPE, e.g.,PEG2000-DMPE), and about 20 mol % to about 40 mol % of one or moresterol derivatives (e.g., cholesterol).

The RNA-binding agent(s) of a lipid particle can include a combinationof any useful lipids and conjugates. In particular embodiments, thecontent of the cationic lipid (e.g., DODMA) is from about 10 mol % toabout 40 mol % (e.g., from about 20 mol % to 40 mol %, 20 mol % to 35mol %, 20 mol % to 30 mol %, 15 mol % to 40 mol %, 15 mol % to 35 mol %,15 mol % to 25 mol %, or 15 mol % to 20 mol %). In some embodiments, thePEG-lipid conjugate (e.g., PEG-DSPE, such as PEG2000-DSPE, and/orPEG-DMPE, such as PEG2000-DMPE) is from about 0.5 mol % to about 10 mol% (e.g., from about 0.5 mol % to 1 mol %, 0.5 mol % to 5 mol %, 0.5 mol%, to 10 mol %, 1 mol % to 5 mol %, or 1 mol % to 10 mol %).

The transfection lipid(s) of a lipid particle can include a combinationof any useful lipids and conjugates. In particular embodiments, thecontent of one or more compounds of Table 1 (e.g., L-6, -30, or any inTable 1) is from about 10 mol % to about 40 mol % (e.g., from about 10mol % to 20 mol %, 10 mol % to 30 mol %, 10 mol % to 35 mol %, 15 mol %to 20 mol %, 15 mol % to 25 mol %, 15 mol % to 30 mol %, 15 mol % to 35mol %, 15 mol % to 40 mol %, 20 mol % to 25 mol %, 20 mol % to 30 mol %,20 mol % to 35 mol %, 20 mol % to 40 mol %, 25 mol % to 30 mol %, 25 mol% to 35 mol %, or 25 mol % to 40 mol %). In some embodiments, thecontent of one or more neutral lipids (e.g., DSPC) is about 5 mol % toabout 20 mol % (e.g., from about 5 mol % to 10 mol %, 5 mol % to 15 mol%, 7 mol % to 10 mol %, 7 mol % to 15 mol %, 7 mol % to 20 mol %, 10 mol% to 15 mol %, or 10 mol % to 20 mol %). In some embodiments, thecontent of one or more PEG-lipid conjugates (e.g., PEG-DSPE, such asPEG2000-DSPE, and/or PEG-DMPE, such as PEG2000-DMPE) is about 0.5 mol %to about 10 mol % (e.g., from about 0.5 mol % to 1 mol %, 0.5 mol % to 5mol %, 0.5 mol %, to 10 mol %, 1 mol % to 5 mol %, or 1 mol % to 10 mol%). In some embodiments, the content of one or more sterol derivatives(e.g., cholesterol) is about 20 mol % to about 40 mol % (e.g., fromabout 20 mol % to 25 mol %, 20 mol % to 30 mol %, 20 mol % to 35 mol %,20 mol % to 40 mol %, 25 mol % to 30 mol %, 25 mol % to 35 mol %, or 25mol % to 40 mol %).

In other embodiments, compounds selected from Table 1 are used in theformulation of the RNA-binding agent(s) (e.g., about 25.9 mol % of L-6,L-30, L-48, or L-49). In particular embodiments, the compound selectedfrom Table 1 used in the formulation of the RNA-binding agent(s) isdifferent from the compound (optionally from Table 1) used in theformulation of the transfection lipid(s) (e.g., 25.9 mol % L-48 as theRNA-binding agent, and 21.6 mol % L-30 as the transfection lipid). Insome embodiments of the formulation, the one or more RNA-binding agentsform an internal aggregate, and the one or more transfection lipids forman external, aggregate surface. In particular embodiments, the external,aggregate surface is not a membrane, a lipid bilayer, and/or amultilamellar layer.

The formulation can also include any useful amount of one or morePEG-lipid conjugates. In some embodiments, the content of the PEG-lipidconjugate in the formulation is from about 1 mol % and about 20 mol %(e.g., from about 1 mol % to about 2 mol %, from about 2 mol % to about4 mol %, from about 2 mol % to about 7 mol %, from about 4 mol % toabout 8 mol %, from about 8 mol % to about 12 mol %, from about 12 mol %to about 16 mol %, or from about 16 mol % to about 20 mol %). In otherembodiments, the content of PEG-lipid conjugate is about 7 mol %, 6 mol%, 3.0 mol %, or 2.5 mol %. Moreover, the PEG-lipid content may bevaried from about 1 mol % to about 20 mol %, by appropriate adjustmentof the content of either DSPC or cholesterol, or both. The PEG-lipid maybe varied by using C14:0 (as in Table 4, e.g., PEG-DSPE or PEG-DMPE,etc.), C16 (PEG-DPPE, PEG-DPG, etc.), C18:0 (PEG-DSPE, PEG-DSG, etc.),or C18:1 (PEG-DOPE, PEG-DOG, etc.). Furthermore, different molecularweight PEG moieties can be used (PEG2000, PEG3400, PEG5000, etc.). Inparticular embodiments, mixed PEG-conjugates are used, as describedherein. In particular embodiments, PEG2000-DSPE is used. In particularembodiments, PEG2000-DMPE is used.

Formulations with RNAi Agents

The processes of the invention can be used to produce a particle and/orformulation containing an RNAi agent by any of the methods describedherein. For example, see: Judge et al., J. Clin. Invest. 119(3):661,2009; Noble et al., Cancer Chemother. Pharmacol. 64(4):741, 2009; Abramset al., Mol. Ther. 18(1):171, 2009; Yagi et al., Cancer Res.69(16):6531, 2009; Ko et al., J. Control. Release 133(2):132, 2009;Mangala et al., Methods Mol. Biol. 555:29, 2009, which are herebyincorporated by reference.

The particle and/or formulation can include an RNAi agent and a lipidmolecule and/or one or more components in any useful ratio. Exemplaryratios include from a (w/w) ratio of from about 1:10 to about 1:100(w/w) (e.g., from about 1:10 to about 1:50, e.g., about 1:20) of RNAiagent:total lipid ratio, where the total lipid ratio is the weight ofthe combination of one or more lipid molecules (e.g., cationic, anionic,or neutral lipids) and one or more components (e.g., sterol derivatives,PEG-lipid conjugates, polyamide-lipid conjugates, gangliosides,antioxidants, surfactants, amphiphilic agents, or salts). In oneembodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid todsRNA ratio) will be in the range of from about 1:1 to about 50:1, fromabout 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 toabout 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

The particle and/or formulation can include an RNAi agent in a doseranging from about 1 mg/kg to about 10 mg/kg of any RNAi agent describedhere. Exemplary doses include 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, and 10 mg/kg of an RNAi agentin the particle or formulation.

Methods of Preparing Formulations

The particles of the invention can be prepared with a variety of usefulprocesses. In one exemplary procedure, the components of the particlesof the invention (e.g., one or more lipids) are dissolved in a solvent(e.g., an aqueous solvent, a non-aqueous solvent, or solvent mixturesthereof). Exemplary FDA-approved solvents for use in the processes ofthe invention include acetic acid, acetone, acetonitrile, anisole,benzene, 1-butanol, 2-butanol, butyl acetate, tert-butylmethyl ether,carbon tetrachloride, chlorobenzene, chloroform, cumene, cyclohexane,1,2-dichloroethane, 1,1-dichloroethene, 1,2-dichloroethene,dichloromethane, 1,2-dimethoxyethane, n,n-dimethylacetamide,n,n-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, ethanol,2-ethoxyethanol, ethyl acetate, ethyleneglycol, ethyl ether, ethylformate, formamide, formic acid, heptane, hexane, isobutyl acetate,isopropyl acetate, methanol, 2-methoxyethanol, methyl acetate,3-methyl-1-butanol, methylbutyl ketone, methylcyclohexane, methylethylketone, methylisobutyl ketone, 2-methyl-1-propanol, n-methylpyrrolidone,nitromethane, pentane, 1-pentanol, 1-propanol, 2-propanol, propylacetate, pyridine, sulfolane, tetrahydrofuran, tetralin, toluene,1,1,1-trichloroethane, 1,1,2-trichloroethene, xylene and combinationsthereof. The resultant lipid suspension can be optionally filtered,mixed (e.g., batch mixed, in-line mixed, and/or vortexed), evaporated(e.g., using a nitrogen or argon stream), re-suspended (e.g., in anaqueous solvent, a non-aqueous solvent, or solvent mixtures thereof),freeze-thawed, extruded, and/or sonicated. Furthermore, the lipidsuspension can be optionally processed by combining with any desiredcomponents (e.g., anionic agents (e.g., one or more RNAi agents),RNA-binding agents, transfection lipids, and/or any lipids describedherein) to produce a final suspension. The one or more desiredcomponents can be provided in the same or different solvent as thesuspension. For example, the lipid suspension can be provided in a firstsolvent or solvent system (e.g., an acidic aqueous solution such aswater-HCl, or one or more aqueous or non-aqueous solvent(s), such aswater, water-ethanol, buffer (e.g., phosphate buffered saline (PBS),Hank's balanced salt solution (HBSS), Dulbecco's phosphate-bufferedsaline (DPBS), Earle's balanced salt solution (EBSS), carbonate,lactate, ascorbate, and citrate, such as 5 mM, 10 mM, 50 mM, 75 mM, 100mM, or 150 mM)), physiological osmolality solution (290 mOsm/kg, e.g.,0.9% saline, 5% dextrose, and 10% sucrose), saline, methanol, ethanol,n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, glycerol,ethylene glycol, propylene glycol, polyethylene glycol, chloroform,dichloromethane, hexane, cyclohexane, acetone, ether, diethyl ether,dioxan, isopropyl ether, tetrahydrofuran, or combinations thereof), andthe anionic agent (e.g., RNAi agent) can be provided in a second solventor solvent system e.g., one or more aqueous or non-aqueous solvent(s),such as water, water-HCl, water-ethanol, buffer (e.g., phosphatebuffered saline (PBS), Hank's balanced salt solution (HBSS), Dulbecco'sphosphate-buffered saline (DPBS), Earle's balanced salt solution (EBSS),carbonate, lactate, ascorbate, and citrate, such as 5 mM, 10 mM, 50 mM,75 mM, 100 mM, or 150 mM)), physiological osmolality solution (290mOsm/kg, e.g., 0.9% saline, 5% dextrose, and 10% sucrose), saline,methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol,tert-butanol, glycerol, ethylene glycol, propylene glycol, polyethyleneglycol, chloroform, dichloromethane, hexane, cyclohexane, acetone,ether, diethyl ether, dioxan, isopropyl ether, tetrahydrofuran, orcombinations thereof). Exemplary concentrations of aqueous solventsand/or buffers include from about 4% to about 8% ethanol (e.g., fromabout 4% to 5%, 5% to 6%, 6%, to 7%, or 7% to 8%), from about 10 mM toabout 100 mM citrate (e.g., from about 10 mM to 30 mM, 30 mM to 50 mM,50 mM to 70 mM, 70 mM to 90 mM, or 90 mM to 100 mM). Any of the solventsor solvent systems can include one or more stabilizers, such as anantioxidant, a salt (e.g., sodium chloride), citric acid, ascorbic acid,glycine, cysteine, ethylenediamine tetraacetic acid (EDTA), mannitol,lactose, trehalose, maltose, glycerol, and/or glucose. In furtherexamples, the one or more anionic agents are introduced into a lipidsuspension using a first solvent or solvent system and then followed byaddition of one or more additional lipids (e.g., transfection lipids) ina second solvent or solvent system, where first and second solvents orsolvent systems are the same or different (e.g., the first solvent orsolvent system is any described herein; and the second solvent orsolvent system is any described herein). In particular embodiments, thesecond solvent or solvent system include one or more aqueous ornon-aqueous solvents selected from the group consisting of saline,buffer (e.g., citrate or PBS), water, and ethanol. The final suspensioncan be optionally separated (e.g., by ultracentrifuge), mixed (e.g.,batch mixed, in-line mixed, and/or vortexed), re-suspended, adjusted(e.g., with one or more solvents or buffer systems), sonicated,freeze-thawed, extruded, and/or purified.

Cationic Lipids

One or more cationic lipids can be included in the particles and/orformulations produced by the methods of the invention. In addition tothe compounds of Table 1, other cationic lipids include, but are notlimited to: N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),N,N-distearyl-N,N-dimethylammonium (DDAB),1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, including chiral formsR-DOTAP and S-DOTAP),N-(1-(2,3-dioleyloxyl)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium(DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS),1,2-dioleoyl-3-dimethylammonium propane (DODAP),N,N-dimethyl-(2,3-dioleyloxyl)propylamine (DODMA),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium(DMRIE), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA),1,2-dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (DPePC),distearyldimethylammonium chloride (DSDMA),1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC, e.g., or achloride salt thereof), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine(16:0 EPC, e.g., or a chloride salt thereof),1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC, e.g., or achloride salt thereof), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine(18:1 EPC, e.g., or a chloride salt thereof), dipalmitoylphosphatidylethanolamidospermine (DPPES), dipalmitoyl phosphatidylethanolamido L-lysine (DPPEL),1-[2-dioleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM), (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride))(SAINT), and C12-200, as described in Love et al., Proc Natl Acad SciUSA, 107(5):1864-1869 (2010), which is incorporated herein by reference.

Cationic lipids include those of different chiral forms (e.g., R or Sforms of any cationic lipid described herein) or any salt forms (e.g., achloride, bromide, trifluoroacetate, or methanesulfonate salt of anycationic lipid described herein).

Additionally, a number of commercial preparations of cationic lipids maybe included in the particle and/or formulation. Such commercialpreparations include, but are not limited to: Lipofectamine™ (acombination of DOSPA and DOPE) and Lipofectin® (a combination of DOTMAand DOPE) from Invitrogen Corp.; and Transfectam® (a compositionincluding DOGS) and Transfast™ from Promega Corp.

Anionic Lipids

One or more anionic lipids can be included in the formulation and/orparticles of the methods of the instant invention. Such anionic lipidsinclude, but are not limited to: phosphatidylglycerols (PGs),cardiolipins (CLs), diacylphosphatidylserines (PSs), diacylphosphatidicacids (PAs), phosphatidylinositols (PIs),N-acylphosphatidylethanolamines (NAPEs),N-succinylphosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, andpalmitoyloleoylphosphatidylglycerol (POPG), as well as different chiralforms (e.g., R or S forms), salt forms (e.g., a chloride, bromide,trifluoroacetate, or methanesulfonate salts), and mixtures thereof.

Neutral Lipids

One or more neutral lipids can be included in the formulation and/orparticles of the methods of the instant invention. Such neutral lipidsinclude, but are not limited to: ceramides, sphingomyelin (SM),diacylglycerols (DAGs), 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC, including chiral forms R-DSPC and S-DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dioleoyl-glycero-sn-3-phosphoethanolamine (DOPE),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE),1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), as well as differentchiral forms (e.g., R or S forms), salt forms (e.g., a chloride,bromide, trifluoroacetate, or methanesulfonate salts), and mixturesthereof. Other diacyl-sn-glycero-3-phosphocholine anddiacyl-glycero-sn-3-phosphoethanolamine lipids may also be used in thelipids particles of the invention.

In some embodiments, the neutral lipid component present in theformulation and/or particles comprises one or more phospholipids. Infurther embodiments, the neutral lipid component comprises a mixture ofone or more phospholipids and cholesterol. In some embodiments, theselection of neutral lipids for use in the formulation and/or particlesis guided by consideration of pharmacokinetic and/or pharmacodynamicproperties, e.g., lipid particle size and stability in the bloodstream.

Sterol Derivatives

One or more sterol derivatives can be included in the formulation and/orparticles of the methods of the instant invention. Without wishing to belimited by theory, sterol derivatives can be used to stabilize theformulation/particles and/or increase transfection. Exemplary sterolderivatives include cholesterol, derivatives of cholestanol (e.g.,cholestanone, cholestenone, or coprostanol);3β-[-(N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-cholesterol, e.g., a hydrochloride salt thereof);bis-guanidium-tren-cholesterol (BGTC);(2S,3S)-2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)carbonylamino)ethyl2,3,4,4-tetrahydroxybutanoate (DPC-1);(2S,3S)-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)2,3,4,4-tetrahydroxybutanoate (DPC-2);bis((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)2,3,4-trihydroxypentanedioate (DPC-3); and6-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)oxidophosphoryloxy)-2,3,4,5-tetrahydroxyhexanoate(DPC-4).

PEG-Lipid Conjugates

One or more PEG-lipid conjugates can be included in formulation and/orparticles of the methods of the instant invention. Without wishing to belimited by theory, PEG-lipid conjugates could act in reducingaggregation of lipid vectors. PEG-lipid conjugates are described in U.S.Pat. No. 5,885,613 and U.S. Patent Publication No. 2003/0077829, whichare hereby incorporated by reference.

PEG-lipid conjugates that may be included in the formulation and/orparticles include, but are not limited to:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethyleneglycol) (PEG-DMPE or DMPE-PEG) (e.g.,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethyleneglycol-2000) (PEG-2000-DMPE or DMPE-PEG or DMPE-PEG2k)),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethyleneglycol) (PEG-DPPE or DPPE-PEG),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethyleneglycol) (PEG-DSPE or DSPE-PEG),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethyleneglycol) (PEG-DOPE or DOPE-PEG),1,2-dimyristoyl-sn-glycerol-3-(methoxy-polyethylene glycol) (PEG-DMG orDMG-PEG) (e.g., 1,2-dimyristoyl-sn-glycerol-3-(methoxy-polyethyleneglycol) (PEG-2000-DMG or DMG-PEG or DMG-PEG2k)),1,2-dipalmitoyl-sn-glycerol-3-(methoxy-polyethylene glycol) (PEG-DPG orDPG-PEG), 1,2-distearoyl-sn-glycerol-3-(methoxy-polyethylene glycol)(PEG-DSG or DSG-PEG), 1,2-dioleoyl-sn-glycerol-3-(methoxy-polyethyleneglycol) (PEG-DOG or DOG-PEG), 3-N-[(ω-methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),R-3-[(ω-methoxy poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine(PEG-2000-C-DOMG), and PEG-ceramide conjugates (e.g., PEG-CerC14 orPEG-CerC20, which are described in U.S. Pat. No. 5,820,873, incorporatedherein by reference). Additional PEG-lipid conjugates include PEGconjugated to any lipid described herein, such asphosphatidylethanolamine or ceramide (see, U.S. Pat. Nos. 5,820,873;5,534,499; and 5,885,613, which is incorporated herein by reference),and salt forms of any PEG-lipid conjugates described herein (e.g.,sodium, ammonium, or trimethylammonium salts).

The PEG-lipid conjugate can include one or more various modifications,such as substitutions with any lipid molecule described herein or withPEG moieties of different molecular weights (e.g., from 300 to 5,000daltons). Exemplary substitutions include use of one or more of C14:0(as in Table 4), C16 (PEG-DPPE, PEG-DPG, etc.), C18:0 (PEG-DSPE,PEG-DSG, etc.), or C18:1 (PEG-DOPE, PEG-DOG, etc.) in combination with apolyethyleneglycol moiety (e.g., PEG2000, PEG3400, PEG5000, etc) to forma PEG-lipid conjugate (e.g., mPEG2000-DMG). Examples of PEG moietieswith various molecular weights include PEG350, PEG550, PEG750, PEG1000,PEG2000, PEG3000, PEG3400, PEG4000, and PEG5000.

Exemplary Lipids

The formulation and/or particles can include one or a combination of anyart-recognized lipids or other associated components, including, e.g.,those described in U.S. Pat. Nos. 6,756,054; 5,976,567; 6,815,432;6,858,225; 6,020,526; 6,638,529; 6,670,393; 6,034,135; 5,958,901;6,172,049; 8,324,366; 8,158,601; 8,034,376; 8,329,070; 7,901,708;8,283,333; 8,236,943; 8,188,263; 8,101,741; 8,058,069; 7,982,027;7,803,397; 7,915,399; 7,807,815; 7,799,565; 7,745,651; 6,841,537;6,410,328; 7,811,602; 7,244,448; and 8,227,443, as well as applicationNos. US 2012/0294905; US 2012/0244207; US 2012/0046478; US 2012/0183602;US 2012/0128760; US 2012/0101148; US 2009/0163705; US 2012/0016006; US2003/0077829; WO 2010/088537; WO 2010/036962; US 2012/0095075; US2012/0058144; US 2012/0027796; US 2011/0311583; US 2012/0027803; WO2010/048536; WO 2011/038031; WO 2009/132131; WO 2009/100351; WO2004/064737; WO 2004/030634; WO 2011/071860; WO 2013/013017; WO2013/013013; WO 2010/057217; WO 2010/036962; WO 2011/153493; WO2011/075656; WO 2010/144740; WO 2009/086558; WO 2010/054405; WO2010/054401; WO 2010/054384; US 2011/0086826; US 2012/0225434; US2011/0117125; WO 2009/086558; US 2011/0256175; WO 2010/042877; US2010/0041152; US 2009/0285878; WO 2009/108235; WO 2009/108235; US2011/0216622; US 2004/0142025; WO 2004/002453; US 2012/0202871; US2011/0076335; WO 2011/000106; WO 2011/000107; US 2011/0195127; WO2011/000108; US 2011/0178155; WO 2009/129319; US 2012/0328668; US2009/0270481; US 2007/0135372; WO 2007/051303; US 2012/0183581; US2010/0130588; US 2009/0291131; WO 2009/127060; WO 2009/082817; US2012/0058188; US 2011/0091525; US 2006/0240093; US 2005/0175682; US2005/0064595; WO 2005/007196; WO 2005/026372; WO 2005/007196; US2011/0224418; US 2008/0249046; US 2006/0051405; US 2006/0025366; WO2006/007712; WO 2006/002538; WO 2006/007712; US 2011/0262527; US2011/0060032; US 2006/0083780; US 2006/0008910; WO 2005/120152; WO2005/121348; WO 2005/120152; US 2005/0118253; US 2013/0022649; WO2011/066651; US 2012/0172411; US 2011/0313017; US 2011/0201667; WO2011/011447; US 2011/0189300; US 2006/0134189; WO 2006/053430; US2011/0177131; US 2007/0135370; WO 2007/048046; US 2011/0071208; US2009/0149403; US 2008/0171716; WO 2008/019486; US 2007/0218122; WO2007/056861; US 2007/0054873; US 2007/0042031; WO 2007/012191; WO2002/088370; US 2003/0108886; WO 2002/088370; WO 2002/087541; WO2011/038160; WO 2010/083615; WO 2011/141705; WO 2011/141704; WO2012/000104; WO 2011/141703; WO 2010/105372; and WO 2006/074546.

Other Components

The formulation and/or particles can include any other component to aidin stabilizing the lipid vector, reducing aggregation of lipid vectors,and/or delivering a therapeutic agent (e.g., an RNAi agent). Exemplarycomponents include polyamide-lipid conjugates (ATTA-lipids) based onω-amino (oligoethyleneglycol) alkanoic acid monomers, such as thosedescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, which isincorporated herein by reference; gangliosides (e.g., asialogangliosideGM1 or GM2; disialoganglioside GD1a, GD1a-NAcGal, GD1-b, GD2, or GD3;globoside, monosialoganglioside GM1, GM2, or GM3, tetrasialogangliosideGQ1b, and trisialoganglioside GT1a or GT1b); antioxidants (e.g.,α-tocopherol or β-hydroxytoluidine); one or more surfactants (e.g.,sorbitan monopalmitate or sorbitan monopalmitate, oily sucrose esters,polyoxyethylene sorbitane fatty acid esters, polyoxyethylene sorbitolfatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylenealkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene-polypropoxyalkyl ethers, block polymers and cetyl ether, as well as polyoxyethylenecastor oil or hydrogenated castor oil derivatives and polyglycerinefatty acid esters, such as Pluronic®, Poloxamer®, Span®, Tween®,Polysorbate®, Tyloxapol®, Emulphor®, or Cremophor® (e.g., Cremophor® ELhaving a major component of glycerol-polyethyleneglycol ricinoleate withfatty acid esters of polyethylene glycol); one or more amphiphilicagents (e.g., vegetable oils, such as soybean oil, safflower oil, oliveoil, sesame oil, borage oil, castor oil, and cottonseed oil; mineraloils and marine oils, hydrogenated and/or fractionated triglyceridesfrom such sources; medium chain triglycerides (MCT-oils, e.g.,Miglyol®), and various synthetic or semisynthetic mono-, di- ortriglycerides, such as the defined nonpolar lipids disclosed in WO92/05571, as well as acetylated monoglycerides, or alkyl esters of fattyacids, such isopropyl myristate, ethyl oleate (see EP 0 353 267) orfatty acid alcohols, such as oleyl alcohol, cetyl alcohol); and one ormore salts, such as any salt described herein. Typically, theconcentration of the lipid component selected to reduce aggregation isabout 1 mol % to 15 mol %.

Lipid Vectors

The formulation and/or particles of the methods of the invention caninclude one or more compounds selected from Table 1, and/or anylipid-based composition capable of transporting a therapeutic agent(e.g., an anionic agent, such as an RNAi agent). Exemplary lipid-basedcompositions include one or more lipid molecules (e.g., compounds ofTable 1, cationic lipids, anionic lipids, or neutral lipids) and/or oneor more components (e.g., sterol derivatives and/or PEG-lipidconjugates).

Lipid vectors can be formed using any biocompatible lipid or combinationof lipids capable for forming a lipid vector (e.g., liposomes,lipoplexes, and micelles). Encapsulation of a therapeutic agent into alipid vector can protect the agent from damage or degradation orfacilitate its entry into a cell. Lipid vectors, as a result of chargeinteractions (e.g., a cationic lipid vector and anionic cell membrane),interact and fuse with the cell membrane, thus releasing the agent intothe cytoplasm. A liposome is a bilayered vesicle comprising one or moreof compounds of the invention, lipid molecules, and/or components. Alipid nanoparticle is a liposome ranging in size from about 1 nm toabout 1,000 nm. A lipoplex is a liposome formed with cationic lipidmolecules to impart an overall positive charge to the liposome. Amicelle is vesicle with a single layer of lipid molecules.

Liposomes

In certain embodiments, the lipid vector is a liposome. Typically, thelipids used are capable of forming a bilayer and are cationic. Classesof suitable lipid molecules include phospholipids (e.g.,phosphotidylcholine), fatty acids, glycolipids, ceramides, glycerides,and cholesterols, or any combination thereof. Alternatively or inaddition, the lipid vector can include neutral lipids (e.g.,dioleoylphosphatidyl ethanolamine (DOPE)). Other lipids that can formlipid vectors are known in the art and described herein.

As used herein, a “lipid molecule” is a molecule with a hydrophobic headmoiety and a hydrophilic tail moiety and may be capable of formingliposomes, including a compound of Table 1 or any cationic, neutral, oranionic lipid described herein. The lipid molecule can optionally bemodified to include hydrophilic polymer groups. Examples of such lipidmolecules include1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG2000-DSPE), e.g., an ammonium salt thereof) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000] (PEG2000-DSPE carboxy).

Examples of lipid molecules include natural lipids, such as cardiolipin(CL), phosphatidic acid (PA), phosphatidylcholine (PC),lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE),phosphatidylglycerol (PG), phosphatidylinositol (PI), andphosphatidylserine (PS); lipid mixtures, such as lechitin;sphingolipids, such as sphingosine, ceramide, sphingomyelin,cerebrosides, sulfatides, gangliosides, and phytosphingosine; cationiclipids, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecylammonium bromide (DDAB),3-β-[N—(N′,N′-dimethylaminoethane)carbamoly]cholesterol (DC-Chol),N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE), N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), and1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA);phosphatidylcholines, such as1,2-dilauroyl-sn-glycero-3-ethylphosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC);phosphoethanolamines, such as1,2-dibutyryl-sn-glycero-3-phosphoethanolamine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl); phosphatidicacids, such as dicetyl phosphate (DCP),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dioleoyl-sn-glycero-3-phosphate; phosphatidylglycerols, such asdipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol(DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol), and1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol);phosphatidylserines, such as1,2-dimyristoyl-sn-glycero-3-phospho-L-serine,1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, and1,2-dioleoyl-sn-glycero-3-phospho-L-serine; cardiolipins, such as1′,3′-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol; andPEG-lipid conjugates, such as1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-750],1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000],1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000], and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000].

Compounds such as those of Table 1 can be combined with any useful lipidcomposition, including commercially available lipid compositions.Examples of such compositions include Lipofectamine™ (a combination ofDOSPA and DOPE) and Lipofectin® (a combination of DOTMA and DOPE) fromInvitrogen Corp.; Transfectam® (a composition including DOGS) andTransfast™ from Promega Corp.; NeuroPORTER™ and Escort™ fromSigma-Aldrich Co.; FuGENE® 6 from Roche; and LipoTAXI® from Strategene.Known lipid compositions include the Trojan Horse Lipsome technology, asdescribed in Boado, Pharm. Res. 24:1772-1787 (2007).

The liposomes can also include other components that aid in theformation or stability of liposomes. Examples of components includecholesterol, antioxidants (e.g., α-tocopherol or β-hydroxytoluidine),surfactants, and salts.

The liposome can be of any useful combination comprising lipidmolecules, including, e.g., one or more compounds of Table 1 and otherlipid components that aid in the formation or stability of liposomes. Aperson of skill in that art will know how to optimize the combinationthat favor encapsulation of a particular agent, stability of theliposome, scaled-up reaction conditions, or any other pertinent factors.Exemplary combinations are described in Boado, Pharm. Res. 24:1772-1787(2007).

Producing liposomes typically occur through a general two-step process.In the first step, the lipids and lipid components are mixed in avolatile organic solvent or mixtures of solvents to ensure a homogenousmixture of lipids. Examples of solvents include chloroform, methanol,cyclohexane, and t-butanol. The solvent is then removed to form a drylipid mixture in a film, powder, or pellet. The solvent can also beremoved by using any known analytical techniques, such as by usingnitrogen, rotary evaporation, spray drying, lyophilization, andvacuum-drying.

In the second step, the dry lipid mixture is hydrated with an aqueoussolution to form liposomes. The agent can be added to the aqueoussolution, which results in the formation of liposomes with encapsulatedagent. Alternatively, the liposomes are first formed with a firstaqueous solution and then exposed to another aqueous solution containingthe agent. Encapsulation of the agent can be promoted by any knowntechnique, such as by repeat freeze-thaw cycles, sonication, or mixing.A further example of this approach is described in Boado, Pharm. Res.24:1772-1787 (2007). Alternatively, the agent is coupled to ahydrophobic moiety (e.g., cholesterol) to produce a lipophilicderivative and the lipophilic derivative is used with other lipidmolecules to form liposomes.

During the second step, the dry lipid mixture may or may not contain thepolypeptide-lipid conjugate. The process can optionally include variousadditional steps, including heating the aqueous solution past the phasetransition temperature of the lipid molecules before adding it to thedry lipid mixture, where particular ranges of temperatures include fromabout 40° C. to about 70° C.; incubating the combination of the drylipid mixture and the aqueous solution, where particular time rangesinclude from about 30 minutes to about 2 hours; mixing of the dry lipidmixture and the aqueous solution during incubation, such as by vortexmixing, shaking, stirring, or agitation; addition of nonelectrolytes tothe aqueous solution to ensure physiological osmolality, such as asolution of 0.9% saline, 5% dextrose, and 10% sucrose; disruption oflarge multilamellar vesicles, such as by extrusion or sonication; andadditional incubation of the pre-formed liposomes with polypeptide-lipidconjugate, where the dry lipid mixture did not contain the lipidmolecules. One of skill in the art will be able to identify theparticular temperature and incubation times during this hydration stepto ensure incorporation of the derivatized lipid molecule into theliposomes or to obtain stable liposomes.

Lipid compounds such as those of Table 1 can be added at any point inthe process of forming liposomes. In one example, the compound is addedto the lipids and lipid components during the formation of the dry lipidmixture. In another example, the compound is added to liposomes that arepre-formed with a dry lipid mixture containing the lipids and lipidcomponents. In yet another example, micelles are formed with thecompound, liposomes are formed with a dry lipid mixture containinglipids and lipid components, and then the micelles and liposomes areincubated together. The aqueous solution can include additionalcomponents to stabilize the agent or the liposome, such as buffers,salts, chelating agents, saline, dextrose, sucrose, etc.

In one example of this procedure, a dry film composed of the lipidmixture is hydrated with an aqueous solution containing an agent. Thismixture is first heated to 50° C. for 30 minutes and then cooled to roomtemperature. Next, the mixture is transferred onto a dry film containingthe polypeptide-lipid conjugate. The mixture is then incubated at 37° C.for two hours to incorporate the polypeptide-lipid conjugate into theliposomes containing the agent. See, e.g., Zhang et al., J. Control.Release 112:229-239 (2006).

Lipid Particles Having a Vesicle Structure

In certain embodiments, the lipid particle comprises a cationic lipid(e.g., DODMA, DOTMA, and/or an amino-amine lipid, amino-amide lipid, orother such lipid, e.g., of Table 1) and an anionic agent (e.g., an RNAiagent), as well as a neutral or zwitterionic lipid, a PEG-lipidconjugate, and, optionally, cholesterol.

Lipid Particles Having One or More RNA-Binding Agents and One or MoreTransfection Lipids

Lipid particles also include those having one or more RNA-binding agentsand one or more transfection lipids. In one embodiment, the one or moreRNA-binding agents form an internal aggregate, and the one or moretransfection lipids form an external, aggregate surface. In particularembodiments, the external, aggregate surface is not a membrane, a lipidbilayer, and/or a multilamellar layer. In certain embodiments, the oneor more RNA-binding agents (e.g., lipids) represent about 10-90% of thetotal lipids. In other embodiments, the one or more RNA-binding agents(e.g., lipids) represent about 50% of the total lipid. In otherembodiments, the one or more RNA-binding agents (e.g., lipids) representabout 30% of the total lipid. In certain embodiments, thecomplex/aggregate of a nucleic acid agent with one or more RNA-bindingagents of the lipid particle comprises a cationic lipid (e.g., DODMA,DOTMA, and/or an amino-amine lipid or amino-amide lipid, e.g., ofTable 1) and an RNAi agent; and the one or more transfection lipids ofthe lipid particle comprise a neutral or zwitterionic lipid, a PEG-lipidconjugate, and, optionally, cholesterol. In other embodiments, the oneor more transfection lipids of the particle comprise a cationic lipid(e.g., DODMA, DOTMA, an amino-amine lipid, and/or an amino-amide lipid),a neutral lipid, a PEG-lipid conjugate, and, optionally, cholesterol.

Scalable Lipid Particle Manufacturing Process

In certain embodiments, the invention provides processes for particleproduction which improve upon processes previously practiced, with suchimproved processes, for example, allowing for production of largeramounts of lipid particles with little or even no significant loss ofparticle efficacy, as compared to other such processes for making lipidparticles and/or formulations. Without wishing to be bound by theory,the processes of the invention are designed to produce a morehomogeneous population of particle sizes and structures than thoseobtained using alternative processes for particle/formation preparation.Such attributes of the instant invention are believed to result from theorder of addition of components during performance of the processesdisclosed herein specifically, where anionic agent-containing complexesare suspended in an aqueous solution and additional lipids are suspendedin a solvent such as ethanol, addition of the ethanol-containing lipidsolution to the aqueous solution containing the anionic agent complexesresults in less disruption/dissociation of anionic agent complexes thanwhen the aqueous solution containing anionic agent complexes is added tothe ethanol-containing lipid solution. When the latter order of additionis performed (aqueous into ethanol), the initial anionic agent complexesadded to the ethanol-containing lipid solution are exposed to anelevated concentration of ethanol, which then declines over timefollowing further addition of the aqueous solution to the ethanolsolution, ultimately to achieve the final ethanol concentration of themixed solution. Exposure of these initial anionic agent complexes to atransiently high concentration of ethanol is thought to be disruptive tosuch complexes, resulting in greater heterogeneity of particlestructures and sizes within an ultimate particle population that alsopossesses reduced activity and/or potency. In contrast, certain aspectsof the instant invention relate to the surprising identification ofimproved particle population structural and size homogeneity, efficacyand/or potency when the order of addition is such that the ethanolsolution containing additional lipids is added to the anionic agentcomplexes suspended in aqueous solution, which causes the anionic agentcomplexes to be exposed to an initially low and then graduallyincreasing concentration of ethanol (to achieve the same finalconcentration as when the order of addition is reversed), in turnresulting in reduced particle disruption and/or dissociation andimproved particle population homogeneity, efficacy and/or potency.

While differences between methods, e.g., that involve addition ofaqueous complexes to solvent (e.g., ethanol)-suspended lipids and theimproved methods of the instant invention can be modest and/or difficultto detect at small production scales (e.g., preparation of 1 mg ofanionic agent in particles in one mL volume of water), such inventivedifferences become much more pronounced and apparent once productionscale is increased. Exemplary particle production scales for theprocesses of the invention include not only small-scale production(e.g., 1 mg of anionic agent in particles), but also 10 mg or more ofanionic agent in particles, 50 mg or more of anionic agent in particles,100 mg or more of anionic agent in particles, 250 mg or more of anionicagent in particles, 500 mg or more of anionic agent in particles, 1 g ormore of anionic agent in particles, 2 g or more of anionic agent inparticles, 3 g or more of anionic agent in particles, 4 g or more ofanionic agent in particles, 5 g or more of anionic agent in particles,7.5 g or more of anionic agent in particles, 10 g or more of anionicagent in particles, 20 g or more of anionic agent in particles, 40 g ormore of anionic agent in particles, 50 g or more of anionic agent inparticles, 100 g or more of anionic agent in particles, 200 g or more oranionic agent in particles, 300 g or more of anionic agent in particles,400 g or more of anionic agent in particles, 500 g or more of anionicagent in particles, 1 kg or more of anionic agent in particles, 2 kg ormore of anionic agent in particles, 3 kg or more of anionic agent inparticles, 4 kg or more of anionic agent in particles, 5 kg or more ofanionic agent in particles, or 10 kg or more of anionic agent inparticles.

In certain embodiments, particles of the improved processes of theinstant invention possess at least 10% greater total efficacy and/orpotency (per particle quantity and/or volume, etc.) than a correspondingpopulation of particles produced by methods involving addition of theaqueous solution to the solvent (e.g., ethanol) solution. Optionally,particles of the improved processes of the instant invention possess atleast 20% greater, at least 30% greater, at least 40% greater, at least50% greater, at least 60% greater, at least 70% greater, at least 80%greater, at least 90% greater, at least 100% greater, at least 200%greater, at least 500% greater, or at least 1000% greater total efficacyand/or potency (per particle quantity and/or volume, etc.) than acorresponding population of particles produced by methods involvingaddition of the aqueous solution to the solvent (e.g., ethanol)solution. In related embodiments, particles of the improved processes ofthe instant invention reduce anionic agent (e.g., RNAi agent) targetgene expression to at least 10% lower absolute levels than acorresponding population of particles produced by methods involvingaddition of the aqueous solution to the solvent (e.g., ethanol)solution. Optionally, particles of the improved processes of the instantinvention reduce anionic agent (e.g., RNAi agent) target gene expressionto at least 20% lower absolute levels, at least 30% lower absolutelevels, at least 40% lower absolute levels, at least 50% lower absolutelevels, at least 60% lower absolute levels, at least 70% lower absolutelevels, at least 80% lower absolute levels, at least 90% lower absolutelevels, at least 95% lower absolute levels, or 100% lower absolutelevels than a corresponding population of particles produced by methodsinvolving addition of the aqueous solution to the solvent (e.g.,ethanol) solution. Such differences or improvements are commonly bestobserved at high levels of particle production, such as at levels ofabout 10 mg or higher, 20 mg or higher, 50 mg or higher, 100 mg orhigher, 250 mg or higher, 500 mg or higher, 1 g or higher, 2 g orhigher, 3 g or higher, 4 g or higher, 5 g or higher, 7.5 g or higher, 10g or higher, 20 g or higher, 40 g or higher, 50 g or higher, 100 g orhigher, 200 g or more or anionic agent, 300 g or higher, 400 g orhigher, 500 g or higher, 1 kg or higher, 2 kg or higher, 3 kg or higher,4 kg or higher, 5 kg or higher, or 10 kg or higher.

The lipid particles of the processes of the invention typically have amean diameter of from about 30 nm to about 150 nm, from about 40 nm toabout 150 nm, from about 50 nm to about 150 nm, from about 60 nm toabout 130 nm, from about 70 nm to about 110 nm, from about 70 nm toabout 100 nm, from about 80 nm to about 100 nm, from about 90 nm toabout 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm, and are substantially non-toxic. In addition,nucleic acids, when present in the lipid particles of the processes ofthe present invention, are resistant in aqueous solution to degradationwith a nuclease. Nucleic acid-lipid particles and certain methods ofpreparation are disclosed in, e.g., U.S. Patent Publication Nos.20040142025 and 20070042031, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

RNAi Agents

RNA interference (RNAi) is a mechanism that inhibits gene expression bycausing the degradation of specific RNA molecules or hindering thetranscription of specific genes. In nature, RNAi targets are often RNAmolecules from viruses and transposons (a form of innate immuneresponse), although it also plays a role in regulating development andgenome maintenance. Key to the mechanism of RNAi are small interferingRNA strands (siRNA), which have sufficiently complementary nucleotidesequences to a targeted messenger RNA (mRNA) molecule. The siRNA directsproteins within the RNAi pathway to the targeted mRNA and degrades them,breaking them down into smaller portions that can no longer betranslated into protein.

The RNAi pathway is initiated by the enzyme Dicer, which cleaves long,double-stranded RNA (dsRNA) molecules into siRNA molecules, typicallyabout 21 to about 23 nucleotides in length and containing about 19 basepair duplexes. One of the two strands of each fragment, known as theguide strand, is then incorporated into the RNA-induced silencingcomplex (RISC) and pairs with complementary sequences. RISC mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex. The outcome of this recognition event ispost-transcriptional gene silencing. This occurs when the guide strandspecifically pairs with a mRNA molecule and induces the degradation byArgonaute, the catalytic component of the RISC complex.

The particles of the methods of the invention can be used to deliver oneor more anionic agents, such as RNAi agents, to a cell in vitro or invivo (e.g., in a subject). RNAi agents can include different types ofdouble-stranded molecules that include either RNA:RNA or RNA:DNAstrands. These agents can be introduced to cells in a variety ofstructures, including a duplex (e.g., with or without overhangs on the3′-terminus), a hairpin loop, or an expression vector that express oneor more polynucleotides capable of forming a double-strandedpolynucleotide alone or in combination with another polynucleotide.Exemplary RNAi agents include siRNA, shRNA, DsiRNA, and miRNA agents,which are described herein. Generally, these agents are about 10 toabout 40 nucleotides in length, and preferred lengths are describedbelow for particular RNAi agents.

Functional gene silencing by an RNAi agent does not necessarily includecomplete inhibition of the targeted gene product. In some cases,marginal decreases in gene product expression caused by an RNAi agentmay translate to significant functional or phenotypic changes in thehost cell, tissue, organ, or animal. Therefore, gene silencing isunderstood to be a functional equivalent and the degree of gene productdegradation to achieve silencing may differ between gene targets or hostcell type.

siRNA

Small interfering RNA (siRNA) are generally double-stranded RNAmolecules of 16 to 30 nucleotides in length (e.g., 18 to 25 nucleotides,e.g., 21 nucleotides) with one or two nucleotide overhangs on the3′-terminii or without any overhangs. A skilled practitioner may varythis sequence length (e.g., to increase or decrease the overall level ofgene silencing). In certain embodiments, the overhangs are UU or dTdT atthe 3′-terminus. Generally, siRNA molecules are completely complementaryto one strand of a target DNA molecule, since even single base pairmismatches have been shown to reduce silencing. In other embodiments,siRNAs may have a modified backbone composition, such as, for example,2′-deoxy- or 2′-O-methyl modifications, or any modifications describedherein.

siRNA refers to a nucleic acid molecule capable of inhibiting ordown-regulating gene expression in a sequence-specific manner; see, forexample, Zamore et al., Cell 101:25 33 (2000); Bass, Nature 411:428-429(2001); Elbashir et al., Nature 411:494-498 (2001); and PCT PublicationNos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO99/07409, and WO 00/44914. Methods of preparing a siRNA molecule for usein gene silencing are described in U.S. Pat. No. 7,078,196, which ishereby incorporated by reference.

shRNA

Short hairpin RNA (shRNA) are single-stranded RNA molecules in which ahairpin loop structure is present, allowing complementary nucleotideswithin the same strand to form intermolecular bonds. shRNA can exhibitreduced sensitivity to nuclease degradation as compared to siRNA. Incertain embodiments, an shRNA have a stem length from 19 to 29nucleotides in length (e.g., 19 to 21 nucleotides or 25 to 29nucleotides). In some embodiments, loop size is between 4 to 23nucleotides in length. shRNA can generally contain one or moremismatches, e.g., G-U mismatches between the two strands of the shRNAstem, without decreasing potency.

DsiRNA

Dicer-substrate RNA (DsiRNA) are double-stranded RNA agents of 25 to 35nucleotides. Agents of such length are believed to be processed by theDicer enzyme of the RNA interference (RNAi) pathway, whereas agentsshorter than 25 nucleotides generally mimic Dicer products and escapeDicer processing. In some embodiments, DsiRNA has a single-strandednucleotide overhang at the 3′-terminal of the antisense or sense strandof 1 to 4 nucleotides (e.g., 1 or 2 nucleotides).

Certain modified structures of DsiRNA agents were previously described,as such as in U.S. Patent Publication No. 2007/0265220, which isincorporated herein by reference. Additional DsiRNA structures andspecific compositions suitable for use in the formulations of theinstant invention are described in U.S. patent application Ser. No.12/586,283; U.S. Patent Publication Nos. 2005/0244858, 2005/0277610,2007/0265220, 2011/0021604, 2010/0173974, 2010/0184841, 2010/0249214,2010/0331389, 2011/0003881, 2011/0059187, 2011/0111056; and PCTPublication Nos. WO 2010/080129, WO 2010/093788, WO 2010/115202, WO2010/115206, WO 2010/141718, WO 2010/141724, WO 2010/141933, WO2011/072292, WO 2011/075188, which are hereby incorporated by reference.Generally, DsiRNA constructs are synthesized using solid phaseoligonucleotide synthesis methods as described for 19-23mer siRNAs (seeU.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; 6,111,086; 6,008,400; and 6,111,086).

miRNA

MicroRNA (miRNA) are single-stranded RNA molecules of 17 to 25nucleotides (e.g., 21 to 23 nucleotides) in length. A skilledpractitioner may vary this sequence length to increase or decrease theoverall level of gene silencing. These agents silence a target gene bybinding complementary sequences on target messenger RNA. As used herein,the term “miRNA precursor” is used to encompass, without limitation,primary RNA transcripts, pri-miRNAs and pre-miRNAs. A “miRNA agent” ofthe invention can include pri-miRNA, pre-miRNA, and/or miRNA (or maturemiRNA). In certain embodiments, an siRNA (e.g., a DsiRNA) of theinvention may present a guide strand that incorporates a miRNA sequence,or is sufficiently homologous to the miRNA sequence to function as saidmiRNA (rendering such siRNA a “miRNA mimetic”).

Antisense Compounds

Exemplary antisense compounds comprise a consecutive nucleoside lengthrange, wherein the upper end of the range is 50 nucleosides and whereinthe lower end of the range is 8 nucleosides. In certain embodiments, theupper end of the range is 35 nucleosides and the lower end of the rangeis 14 nucleosides. In further embodiments, the upper end of the range is24 nucleosides and the lower end of the range is 17 nucleosides. Instill further embodiments, the antisense compound is 20 consecutivenucleosides. Those skilled in the art will readily recognize that theupper end of the range, as disclosed herein comprises 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive nucleosides and thelower end of the range comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 consecutive nucleosides.

Exemplary antisense compounds comprise a stretch of at least 8,optionally at least 12, optionally at least 15 consecutive nucleosidesthat is sufficiently complementary to a target sequence to interferewith transcription, translation, promote degradation (optionallynuclease-mediated degradation) and/or otherwise disrupt the function(e.g., interfere with the function of an otherwise functional targetsequence, e.g., disruption of a promoter, enhancer or other functionalnucleic acid target sequence via an antisense compound-mediatedmechanism) of the target sequence.

Modifications can be made to antisense compounds and may includeconjugate groups attached to one of the termini, selected nucleobasepositions, sugar positions or to one of the internucleoside linkages.Possible modifications include, but are not limited to, 2′-fluoro(2′-F), 2′-OMethyl (2′-OMe), 2′-O-(2-methoxyethyl) (2′-MOE) highaffinity sugar modifications, inverted abasic caps, deoxynucleobases,and bicyclic nucleobase analogs, such as locked nucleic acids (LNA) andethylene-bridged nucleic acids (ENA).

Method of Making RNAi Agents

RNAi agents include at least one antisense nucleotide sequence that isdirected to a target nucleic acid (e.g., a target gene). Antisensenucleotides are single strands of DNA or RNA that are complementary to achosen target sequence. In the case of antisense RNA, they preventtranslation of complementary RNA strands by binding to it. Antisense DNAcan be used to target a specific, complementary (coding or non-coding)RNA. In a particular embodiment, antisense nucleotides contain fromabout 10 to about 40 nucleotides, more preferably about 15 to about 30nucleotides. The antisense nucleotide can have up to 80%, 85%, 90%, 95%,99%, or even 100% complementary to the desired target gene.

Methods of producing antisense and sense nucleotides, as well ascorresponding duplexes or hairpin loops, are known in the art and can bereadily adapted to produce an antisense oligonucleotide that targets anytarget nucleic acid sequence. Antisense nucleotide sequences can beselected to optimize target specificity, such as by analyzing the targetsequence and determining secondary structure, Tm, binding energy, andrelative stability; and/pr to reduce the formation of secondarystructures, such as dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. In some embodiments, highly preferred target regions of the mRNAinclude those regions at or near the AUG translation initiation codonand those sequences that are substantially complementary to 5′ regionsof the mRNA. These secondary structure analyses and target siteselection considerations can be performed, for example, using v.4 of theOLIGO primer analysis software (Molecular Biology Insights) and/or theBLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997). Non-limiting methods for preparing RNAi agentsare described in U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203;6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; 6,111,086;6,008,400; and 6,111,086, which are incorporated herein by reference.

The RNAi agents can have any useful form, such as single-stranded,double-stranded, linear, circular (e.g., a plasmid), nicked circular,coiled, supercoiled, concatemerized, or charged. Additionally,nucleotides may contain 5′ and 3′ sense and antisense strand terminalmodifications and can have blunt or overhanging terminal nucleotides(e.g., UU or TT at the 3′-terminus), or combinations thereof.

Modified nucleic acids, including modified DNA or RNA molecules, may beused in the in place of naturally occurring nucleic acids in thepolynucleotides (e.g., RNAi agents) described herein. Modified nucleicacids can improve the half-life, stability, specificity, delivery,solubility, and nuclease resistance of the polynucleotides describedherein. For example, siRNA agents can be partially or completed composedof nucleotide analogs that confer the beneficial qualities describedabove. As described in Elmen et al. (Nucleic Acids Res. 33:439-447(2005)), synthetic, RNA-like nucleotide analogs (e.g., locked nucleicacids (LNA)) can be used to construct siRNA molecules that exhibitsilencing activity against a target gene product.

The phosphorothioate (PS) backbone modification, where a non-bridgingoxygen in the phosphodiester bond is replaced by sulfur, is one of theearliest and most common means deployed to stabilize nucleic acid drugsagainst nuclease degradation. In general, it appears that PSmodifications can be made extensively to both siRNA strands without muchimpact on activity (Kurreck, Eur. J. Biochem. 270:1628-44 (2003)). Inparticular embodiments, the PS modification is usually restricted to oneor two bases at the 3′ and 5′ ends. The boranophosphate linker can beused to enhance siRNA activity while having low toxicity (Hall et al.,Nucleic Acids Res. 32:5991-6000 (2004)). Other exemplary modificationsto the oligonucleotide backbone include methylphosphonates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates (e.g., 3′-alkylene phosphonate), chiral phosphonates,phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate),aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, and a proteinnucleotide (PNA) backbone having repeating N-(2-aminoethyl)-glycineunits linked by peptide bonds, where representative PNA compoundsinclude, but are not limited to, those disclosed in U.S. Pat. Nos.5,539,082, 5,714,331, and 5,719,262, and Nielsen et al., Science254:1497-1500 (1991).

Other modifications to the backbone include those replacing thephosphorous atom with short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages (e.g., morpholino linkages; siloxane backbones;sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulphamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts).

Certain modified nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention, such as5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil,5-propynylcytosine, and 5-methylcytosine). Exemplary modifiednucleobases include 5-methylcytosine (5-me-C or m5c); 5-hydroxymethylcytosine, xanthine, and hypoxanthine; 2-aminoadenine, 6-methyl, andother alkyl derivatives of adenine and guanine; 2-propyl and other alkylderivatives of adenine and guanine; 2-thiouracil; 2-thiothymine;2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil andcytosine; 6-azo uracil, cytosine, and thymine; 5-uracil (pseudouracil);4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, andother 8-substituted adenines and guanines; 5-halo, particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines;7-methylguanine; 7-methyladenine; 8-azaguanine; 8-azaadenine;7-deazaguanine; 7-deazaadenine; 3-deazaguanine; and 3-deazaadenine.These modified nucleobases may be combined, in particular embodiments,with other modifications, such as any sugar modification describedherein.

Modified oligonucleotides may also contain one or more substituted sugarmoieties, where modifications can be made at any reactive site of theribose ring (e.g., the 2′-OH of the ribose ring), or one or moreuniversal bases. Exemplary modifications include 2′-halo, such as F, Br,or Cl; 2′-O-alkyl, 2′-S-alkyl, or 2′-N-alkyl, such as 2′-OMe;2′-O-(alkyl-O)_(n)-alkyl, such as 2′-O-methoxyethyl (2′-O-MOE),2′-O[(CH₂)_(n)O]_(m)CH₃, 2′-O(CH₂)_(n)OCH₃,2′-O(CH₂)₂ON(CH₃)₂O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, 2′-O(CH₂)_(n)ONH₂, and2′-O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10;2′-O-alkenyl, 2′-S-alkenyl, or 2′-N-alkenyl; 2′-O-alkynyl, 2′-S-alkynyl,or 2′-N-alkynyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl and alkynyl,as well as a bridging modification between the 2′ and 4′ positions ofribose to form a locked nucleic acid (LNA). Exemplary universal basesinclude a heterocyclic moiety located at the 1′ position of a nucleotidesugar moiety in a modified nucleotide, or the equivalent position in anucleotide sugar moiety substitution, such as1-β-D-ribofuranosyl-5-nitroindole and1-β-D-ribofuranosyl-3-nitropyrrole.

In certain embodiments, nucleic acids possessing described forms ofmodification and/or patterns of modification can be employed. Additionaldetail regarding exemplary modifications and modification patterns ofnucleic acids can be found, e.g., in at least the following references:US 2010/0240734; WO 2010/080129; WO 2010/033225; US 2011/0021604; WO2011/075188; WO 2011/072292; WO 2010/141724; WO 2010/141726; WO2010/141933; WO 2010/115202; WO 2008/136902; WO 2011/109294; WO2011/075188; PCT/US11/42810; PCT/US11/42820; U.S. Ser. No. 61/435,304;U.S. Ser. No. 61/478,093; U.S. Ser. No. 61/497,387; U.S. Ser. No.61/529,422; U.S. Pat. No. 7,893,245; WO 2007/051303; and US2010/0184209. Each of the preceding documents is hereby incorporated byreference in its entirety.

RNAi Gene Targets

In certain embodiments, the present invention features the silencing ofa target gene in a diseased tissue or organ by treatment with a particleor formulation, in combination with an RNAi agent. The therapeuticpotential of the present invention is realized when the mRNA moleculesof a specific and targeted gene known or thought to be involved in theestablishment or maintenance of the disease state (e.g., a cancer) aredegraded by the RNAi agent.

Examples of RNAi targets for use with the present invention includedevelopmental proteins, such as adhesion molecules, cyclin kinaseinhibitors, Wnt family members, Pax family members, Winged helix familymembers, Hox family members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors; oncogene-encoded proteins (e.g., ABL1 (UniProtEntry No. P00519, NCBI Gene ID: 25), AR (UniProt Entry No. P10275, NCBIGene ID: 3647), β-Catenin (CTNNB1, UniProt Entry No. P35222, NCBI GeneID: 1499), BCL1 (UniProt Entry No. P24385, NCBI Gene ID: 595), BCL2(UniProt Entry No. P10415, NCBI Gene ID: 596), BCL6 (UniProt Entry No.P41182), CBFA2 (UniProt Entry No. Q01196, NCBI Gene ID: 861), CBL(UniProt Entry No. P22681, NCBI Gene ID: 687), CSF1R (UniProt Entry No.P07333, NCBI Gene ID: 1436), ERBA1 (UniProt Entry No. P10827, NCBI GeneID: 7067), ERBA2 (UniProt Entry No. P10828, NCBI Gene ID: 7068), ERBB(UniProt Entry No. P00533, NCBI Gene ID: 1956), ERBB2 (UniProt Entry No.P04626, NCBI Gene ID: 2064), ERBB3 (UniProt Entry No. P21860, NCBI GeneID: 190151), ERBB4 (UniProt Entry No. Q15303, NCBI Gene ID: 600543),ETS1 (UniProt Entry No. P14921, NCBI Gene ID: 2113), ETS2 (UniProt EntryNo. P15036, NCBI Gene ID: 2114), ETV6 (UniProt Entry No. 41212, NCBIGene ID: 2120), FGR (UniProt Entry No. P09769, NCBI Gene ID: 2268), FOS(UniProt Entry No. P0110, NCBI Gene ID: 2353), FYN (UniProt Entry No.P06241, NCBI Gene ID: 2534), HCR (UniProt Entry No. Q8TD31, NCBI GeneID: 54535), HRAS (UniProt Entry No. P01112, NCBI Gene ID: 3265), JUN(UniProt Entry No. P05412, NCBI Gene ID: 3725), KRAS (UniProt Entry No.P01116, NCBI Gene ID: 3845), LCK (UniProt Entry No. P06239 NCBI Gene ID:3932), LYN (UniProt Entry No. P07948, NCBI Gene ID: 4067), MDM2 (UniProtEntry No. Q00987, NCBI Gene ID: 4193), MLL1 (UniProt Entry No. Q03164,NCBI Gene ID: 4297), MLL2 (UniProt Entry No. 014686, NCBI Gene ID:8085), MLL3 (UniProt Entry No. Q8NEZ4, NCBI Gene ID: 58508), MYB(UniProt Entry No. P10242, NCBI Gene ID: 4602), MYC (UniProt Entry No.P01106, NCBI Gene ID: 4609), MYCL1 (UniProt Entry No. P12524, NCBI GeneID: 4610), MYCN (UniProt Entry No. P04198, NCBI Gene ID: 4613), NRAS(UniProt Entry No. P01111, NCBI Gene ID: 4893), PIM1 (UniProt Entry No.P11309, NCBI Gene ID: 5292), PML (UniProt Entry No. P29890, NCBI GeneID: 5371), RET (UniProt Entry No. P07949, NCBI Gene ID: 5979), SRC(UniProt Entry No. P12931, NCBI Gene ID: 6714), TAL1 (UniProt Entry No.P17542, NCBI Gene ID: 6886), TAL2 (UniProt Entry No. Q16559, NCBI GeneID: 6887), TCL3 (UniProt Entry No. P31314, NCBI Gene ID: 3195), TCL5(UniProt Entry No. P17542, NCBI Gene ID: 6886), and YES (UniProt EntryNo. P07947, NCBI Gene ID: 7525)); tumor suppressor proteins (e.g., BRCA1(UniProt Entry No. P38398, NCBI Gene ID: 672), BRCA2 (UniProt Entry No.P51587, NCBI Gene ID: 675), MADH4 (UniProt Entry No. Q13485, NCBI GeneID: 4089), MCC (UniProt Entry No. P23508, NCBI Gene ID: 4163), NF1(UniProt Entry No. P21359, NCBI Gene ID: 4763), NF2 (UniProt Entry No.P35240, NCBI Gene ID: 4771), RB1 (UniProt Entry No. P06400, NCBI GeneID: 5925), TP53 (UniProt Entry No. P04637, NCBI Gene ID: 7157), PLK1(UniProt Entry No. P53350, NCBI Gene ID: 9606), KIF1-binding protein(UniProt Entry No. Q96EK5, NCBI Gene ID: 9606), and WT1 (UniProt EntryNo. P19544, NCBI Gene ID: 4790)); lipoproteins (e.g., apolipoprotein B(ApoB100, UniProt Entry No. P04114, NCBI Gene ID: 338)); enzymes (e.g.,ACC synthases and oxidases, ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, chalcone synthases,chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNApolymerases, galactosidases, glucanases, glucose oxidases, granule-boundstarch synthases, GTPases, helicases, hernicellulases, integrases,inulinases, invertases, isomerases, kinases (e.g., PLK1 (UniProt EntryNo. P53350, NCBI Gene ID: 9606)), lactases, ligases (e.g., ring finger-and WD repeat-containing protein 2 (RFWD2), also known as COP1),lipases, lipoxygenases, lysozymes, nopaline synthases, octopinesynthases, pectinesterases, peroxidases, phosphatases, phospholipases,phosphorylases, phytases, plant growth regulator synthases,polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, ribulose-1,5-bisphosphatecarboxylase oxygenases (RuBisCos), topoisomerases, transferases, such ashypoxanthine guanine phosphoribosyltransferase 1 (HPRT1), andxylanases).

The liver is one of the most important target tissues for nucleic acidtherapy given its central role in metabolism (e.g., lipoproteinmetabolism in various hypercholesterolemias) and the secretion ofcirculating proteins (e.g., clotting factors in hemophilia). Inaddition, acquired disorders such as chronic hepatitis and cirrhosis arecommon and are also potentially treated by polynucleotide-based livertherapies. A number of diseases or conditions which affect or areaffected by the liver are potentially treated through knockdown(inhibition) of gene expression in the liver. Exemplary liver diseasesand conditions may be selected from the list comprising: liver cancers(including hepatocellular carcinoma, HCC), viral infections (includinghepatitis), metabolic disorders, (including hyperlipidemia anddiabetes), fibrosis, and acute liver injury. Exemplary molecular targetsfor liver therapeutics (e.g., including therapeutics targeted to HCC inparticular) and optionally for therapeutics addressing other targets,diseases and/or disorders, including other cancers include CSN5 (UniProtEntry No. Q92905, NCBI Gene ID: 10987), CDK6 (UniProt Entry No. Q00534,NCBI Gene ID: 1021), ITGB1 (UniProt Entry No. P05556, NCBI Gene ID:3688), MYC (UniProt Entry No. P01106, NCBI Gene ID: 4609), TGFβ1(UniProt Entry No. P01137, NCBI Gene ID: 7040), Cyclin D1 (UniProt EntryNo. Q9H014, NCBI Gene ID: 595), hepcidin (UniProt Entry No. P81172, NCBIGene ID: 57817), PCSK9 (UniProt Entry No. Q8NBP7, NCBI Gene ID: 255738),and transthyretin (TTR, UniProt Entry No. P02766, NCBI Gene ID: 7276),among others.

Particles and/or formulations of the methods of the invention optionallycan be targeted to normal tissues (e.g., normal liver tissue), as wellas to various models (e.g., orthotopic liver models, subcutaneous livermodels, etc.).

One exemplary target for the particles of the processes of the inventionis Apolipoprotein B (ApoB), which is found in various classes oflipoproteins: chylomicrons, very low density lipoproteins (VLDL),intermittent density lipoproteins (IDL), and low density lipoproteins(LDL). ApoB functions as a recognition signal for the cellular bindingand internalization of LDL particles by the ApoB/E receptor. Anaccumulation or overabundance of apolipoprotein B-containinglipoproteins can lead to lipid-related disorders such asatherosclerosis. Formulated therapies that reduce ApoB can be useful fortreating lipid-related disorders. One nucleic acid based therapy, in theform of antisense therapy, has been shown to reduce ApoB levels in mousein vivo, and treatments subsequently reduced serum cholesterol andtriglyceride levels (U.S. Publication No. 2003/0215943). These resultsdemonstrated a moderate downregulation of ApoB and its use as a targetin treating lipid-related disorders.

Another exemplary target for the particles of the processes of theinvention is Protein C, which may be targeted, e.g., for the treatmentof hemophilia.

Lipid-DsiRNA nanoparticles typically form spontaneously upon mixinglipids with DsiRNAs to form a complex. Depending on the desired particlesize distribution, the resultant nanoparticle mixture can be extrudedthrough a polycarbonate membrane (e.g., 100 nm cut-off) using, forexample, a thermobarrel extruder, such as Lipex Extruder (NorthernLipids, Inc). In some cases, the extrusion step can be omitted. Infurther preparation of a particle for use, ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

Formulations of particles are typically characterized by visualinspection. They should be whitish translucent solutions free fromaggregates or sediment. Particle size and particle size distribution oflipid-nanoparticles can be measured by light scattering using, forexample, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should beabout 20-300 nm, such as 40-100 nm in size. The particle sizedistribution should be unimodal. The total DsiRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated DsiRNA can be incubated withan RNA-binding dye, such as Ribogreen (Molecular Probes) in the presenceor absence of a formulation disrupting surfactant, e.g., 0.5%Triton-X100. The total DsiRNA in the formulation can be determined bythe signal from the sample containing the surfactant, relative to astandard curve. The entrapped fraction is determined by subtracting the“free” DsiRNA content (as measured by the signal in the absence ofsurfactant) from the total DsiRNA content. Percent entrapped DsiRNA istypically >85%. For certain formulations, the particle size is at least30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm,at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and atleast 120 nm. The suitable range is typically about at least 50 nm toabout at least 110 nm, about at least 60 nm to about at least 100 nm, orabout at least 80 nm to about at least 90 nm.

Delivery of a Therapeutic Agent

The particles and/or formulations of the processes of the invention maybe used to deliver a therapeutic agent (e.g., anionic agents, such asnucleic acids or RNAi agents) to cells. The agent delivered by theparticles and/or formulations can be used for gene-silencing (e.g., invitro or in vivo in a subject) or to treat or prophylactically treat adisease (e.g., cancer) in a subject.

Delivery of a therapeutic agent may be assessed by using any usefulmethod. For example, delivery with a particle and/or formulationsproduced by the processes of the invention may be assessed by 1)knockdown of a target gene or 2) toxicity or tolerability, as comparedto a control at an equivalent dose. These assessments can be determinedwith any useful combination of lipids in the particle and/orformulation, such as any cationic lipid described herein (e.g., DOTAP,DODMA, DLinDMA, and/or DLin-KC2-DMA), optionally in combination, e.g.,with a compound of Table 1. In particular embodiments, an improvement ofdelivery of a therapeutic agent (e.g., anionic agent, such as an RNAiagent) is observed when using a process of the invention, where theimprovement is more than 25% (e.g., more than a 2-fold, 5-fold, 10-fold,100-fold, or 1000-fold improvement in delivery), as compared to acontrol.

Delivery of RNAi Agents

RNAi silencing can be used in a wide variety of cells, where HeLa S3,COST, 293, NIH/3T3, A549, HT-29, CHO-KI and MCF-7 cell lines are amongthose susceptible to some level of siRNA silencing. Furthermore,suppression in mammalian cells can occur at the RNA level withspecificity for the targeted genes, where a strong correlation betweenRNA and protein suppression has been observed. Accordingly, theparticles produced by the processes of the invention, and formulationsthereof, may be used to deliver an RNAi agent to one or more cells(e.g., in vitro or in vivo). Exemplary RNAi agents include siRNA, shRNA,dsRNA, miRNA, and DsiRNA agents, as described herein.

In Vitro Target Knockdown

Delivery of a RNAi agent can be assessed by any useful method. Forexample, formulations including a therapeutic agent can be transfectedin vitro in cell culture models (e.g., HeLa cells), where end pointmeasurements include, but are not limited to, one or more of thefollowing: (i) mRNA quantification using qPCR; (ii) proteinquantification using Western blot; (iii) labeled cell internalization ofthe anionic agent and/or a cationic lipid of a particle made by theprocesses of the invention. Uptake or delivery may be assessed for boththe extent and duration of the above-mentioned end points. Prior todelivery, the formulation may be diluted in cell culture media at roomtemperature for about 30 minutes, and the final concentration can bevaried from 0 to 50 nM of the anionic agent or of one or more lipids orother particle and/or formulation components in dose-responseexperiments. For time-course experiments, an optimum concentration fromthe dose-experiment may be studied for various incubation times, e.g.,30 minutes to 7 days.

The functionality of anionic agent and lipid formulations may also betested by differentially labeling the lipid compound and the therapeuticagent with fluorescent tags and performing fluorescent colocalizationstudies. The ability of the compounds of the invention to deliveranionic agents and/or an attached fluorescent label may be assessed bothby measuring the total fluorescence inside the cell and by measuringfluorescence that is not stably associated with endosomal or lysosomalcompartments (to function, therapeutic agents that trigger RNAi arerequired not only to reach inside the cell, but also to reach thecytoplasm of the cell). Performance of fluorescence localization andcellular trafficking studies has been described in the art (Lu, et al.,Mol. Pharm. 6(3):763, 2009; McNaughton et al., Proc. Natl. Acad. Sci.U.S.A. 106(15):6111, 2009).

Delivery to Particular Target Cell Types and Target Tissues

The particles made by the processes of the invention can be used todeliver therapeutic agents (e.g., anionic agents) to various organs andtissues to treat various diseases. Exemplary targeted tissues or organsinclude, but are not limited to, liver, pancreas, lung, prostate,kidney, bone marrow, spleen, thymus, lymph node, brain, spinal cord,heart, skeletal muscle, skin, oral mucosa, esophagus, stomach, ileum,small intestine, colon, bladder, cervix, ovary, testis, mammary gland,adrenal gland, adipose tissue (white and/or brown), blood (e.g.,hematopoietic cells, such as human hematopoietic progenitor cells, humanhematopoietic stem cells, CD34+ cells, CD4+ cells), lymphocytes andother blood lineage cells.

Cancer Therapy

The particles produced by the processes of the invention can be used todeliver one or more therapeutic agents (e.g., RNAi agents) to a subjecthaving cancer or at risk of developing a cancer (e.g., an increased riskof at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).Exemplary cancers include liver cancer (e.g., hepatocellular carcinoma,hepatoblastoma, cholangiocarcinoma, angiosarcoma, or hemangiosarcoma) orneuroblastoma. Exemplary neoplastic diseases and associatedcomplications include, but are not limited to, carcinomas (e.g., lung,breast, pancreatic, colon, hepatocellular, renal, female genital tract,squamous cell, carcinoma in situ), lymphoma (e.g., histiocytic lymphoma,non-Hodgkin's lymphoma), MEN2 syndromes, neurofibromatosis (includingSchwann cell neoplasia), myelodysplastic syndrome, leukemia, tumorangiogenesis, cancers of the thyroid, liver, bone, skin, brain, centralnervous system, pancreas, lung (e.g., small cell lung cancer, non smallcell lung cancer (NSCLC)), breast, colon, bladder, prostate,gastrointestinal tract, endometrium, fallopian tube, testes and ovary,gastrointestinal stromal tumors (GISTs), prostate tumors, mast celltumors (including canine mast cell tumors), acute myeloid myelofibrosis,leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chroniclymphocytic leukemia, multiple myeloma, melanoma, mastocytosis, gliomas,glioblastoma, astrocytoma, neuroblastoma, sarcomas (e.g., sarcomas ofneuroectodermal origin or leiomyosarcoma), metastasis of tumors to othertissues, and chemotherapy-induced hypoxia.

Administration and Dosage

The present invention also relates to processes for production ofpharmaceutical compositions that contain a compound or a therapeuticallyeffective amount of a composition, such as a formulation including atherapeutic agent (e.g., an RNAi agent). The composition can beformulated for use in a variety of drug delivery systems. One or morephysiologically acceptable excipients or carriers can also be includedin the composition for proper formulation. Suitable formulations for usein the present invention are found in Remington's PharmaceuticalSciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985.For a brief review of methods for drug delivery, see, e.g., Langer,Science 249:1527-1533, 1990.

The pharmaceutical compositions are intended for parenteral, intranasal,topical, oral, or local administration, such as by a transdermal means,for prophylactic and/or therapeutic treatment. The pharmaceuticalcompositions can be administered parenterally (e.g., by intravenous,intramuscular, or subcutaneous injection), or by oral ingestion, or bytopical application or intraarticular injection at areas affected by thevascular or cancer condition. Additional routes of administrationinclude intravascular, intra-arterial, intratumor, intraperitoneal,intraventricular, intraepidural, as well as nasal, ophthalmic,intrascleral, intraorbital, rectal, topical, or aerosol inhalationadministration. Sustained release administration is also specificallyincluded in the invention, by such means as depot injections or erodibleimplants or components. Thus, the invention provides compositions forparenteral administration that comprise the above mention agentsdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS, and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents and the like. The invention also providescompositions for oral delivery, which may contain inert ingredients suchas binders or fillers for the formulation of a tablet, a capsule, andthe like. Furthermore, this invention provides compositions for localadministration, which may contain inert ingredients such as solvents oremulsifiers for the formulation of a cream, an ointment, and the like.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above mentioned agent or agents, suchas in a sealed package of tablets or capsules. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The compositions containing an effective amount can be administered forprophylactic or therapeutic treatments. In prophylactic applications,compositions can be administered to a patient with a clinicallydetermined predisposition or increased susceptibility to development ofa tumor or cancer. Compositions of the invention can be administered tothe patient (e.g., a human) in an amount sufficient to delay, reduce, orpreferably prevent the onset of clinical disease or tumorigenesis. Intherapeutic applications, compositions are administered to a patient(e.g., a human) already suffering from a cancer in an amount sufficientto cure or at least partially arrest the symptoms of the condition andits complications. An amount adequate to accomplish this purpose isdefined as a “therapeutically effective dose,” an amount of a compoundsufficient to substantially improve some symptom associated with adisease or a medical condition. For example, in the treatment of cancer,an agent or compound which decreases, prevents, delays, suppresses, orarrests any symptom of the disease or condition would be therapeuticallyeffective. A therapeutically effective amount of an agent or compound isnot required to cure a disease or condition but will provide a treatmentfor a disease or condition such that the onset of the disease orcondition is delayed, hindered, or prevented, or the disease orcondition symptoms are ameliorated, or the term of the disease orcondition is changed or, for example, is less severe or recovery isaccelerated in an individual.

Amounts effective for this use may depend on the severity of the diseaseor condition and the weight and general state of the patient, butgenerally range from about 0.5 mg to about 3000 mg of the agent oragents per dose per patient. Suitable regimes for initial administrationand booster administrations are typified by an initial administrationfollowed by repeated doses at one or more hourly, daily, weekly, ormonthly intervals by a subsequent administration. The total effectiveamount of an agent present in the compositions of the invention can beadministered to a mammal as a single dose, either as a bolus or byinfusion over a relatively short period of time, or can be administeredusing a fractionated treatment protocol, in which multiple doses areadministered over a more prolonged period of time (e.g., a dose every4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2 weeks, once amonth). Alternatively, continuous intravenous infusion sufficient tomaintain therapeutically effective concentrations in the blood arecontemplated.

The therapeutically effective amount of one or more agents presentwithin the compositions of the invention and used in the methods of thisinvention applied to mammals (e.g., humans) can be determined by theordinarily-skilled artisan with consideration of individual differencesin age, weight, and the condition of the mammal. The agents of theinvention are administered to a subject (e.g., a mammal, such as ahuman) in an effective amount, which is an amount that produces adesirable result in a treated subject (e.g., the slowing or remission ofa cancer or neurodegenerative disorder). Such therapeutically effectiveamounts can be determined empirically by those of skill in the art.

The patient may also receive an agent in the range of about 0.1 to 3,000mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 ormore times per week), 0.1 to 2,500 (e.g., 2,000, 1,500, 1,000, 500, 100,10, 1, 0.5, or 0.1) mg dose per week. A patient may also receive anagent of the composition in the range of 0.1 to 3,000 mg per dose onceevery two or three weeks.

The amount (dose) of formulation and agent (e.g., DsiRNA) that is to beadministered can be determined empirically. In certain embodiments,effective knockdown of gene expression is observed using 0.0001-10 mg/kganimal weight of nucleic acid agent and 0.001-200 mg/kg animal weightdelivery formulation. An exemplary amount in mice is 0.1-5 mg/kg nucleicacid agent and 0.7-100 mg/kg delivery formulation. Optionally, about1-50 mg/kg delivery formulation is administered. The amount of agent(e.g., DsiRNA) is easily increased because it is typically not toxic inlarger doses.

In certain embodiments, doses can be administered daily over a period ofdays, weeks, or longer (e.g., between one and 28 days or more), or onlyonce, or at other intervals, depending upon, e.g., acute versus chronicindications, etc.

Single or multiple administrations of the compositions of the inventioncomprising an effective amount can be carried out with dose levels andpattern being selected by the treating physician. The dose andadministration schedule can be determined and adjusted based on theseverity of the disease or condition in the patient, which may bemonitored throughout the course of treatment according to the methodscommonly practiced by clinicians or those described herein.

The compounds and formulations of the present invention may be used incombination with either conventional methods of treatment or therapy ormay be used separately from conventional methods of treatment ortherapy. When the compounds and formulations of this invention areadministered in combination therapies with other agents, they may beadministered sequentially or concurrently to an individual.Alternatively, pharmaceutical compositions according to the presentinvention include a combination of a compound or formulation of thepresent invention in association with a pharmaceutically acceptableexcipient, as described herein, and another therapeutic or prophylacticagent known in the art.

The formulated agents can be packaged together as a kit. Non-limitingexamples include kits that contain, e.g., two pills, a pill and apowder, a suppository and a liquid in a vial, two topical creams, etc.The kit can include optional components that aid in the administrationof the unit dose to patients, such as vials for reconstituting powderforms, syringes for injection, customized IV delivery systems, inhalers,etc. Additionally, the unit dose kit can contain instructions forpreparation and administration of the compositions. The kit may bemanufactured as a single use unit dose for one patient, multiple usesfor a particular patient (at a constant dose or in which the individualcompounds may vary in potency as therapy progresses); or the kit maycontain multiple doses suitable for administration to multiple patients(“bulk packaging”). The kit components may be assembled in cartons,blister packs, bottles, tubes, and the like.

EXAMPLES Example 1 Process for Production of Anionic Agent-ContainingParticles (Process 2141)

HPRT1 & MYC DsiRNAs

Particles were prepared as described below with a cationic lipid(DODMA), a neutral lipid (DSPC), a PEG-lipid conjugate (PEG-DMPE andPEG-DMG), and cholesterol with an RNAi agent (DsiRNA for HPRT1 or MYC),having one of the following structures:

HPRT 1: (SEQ ID NO: 1) 5′-GCCAGACUUUGUUGGAUUUGAAAtt (SEQ ID NO: 2)3′-UUCGGUCUGAAACAACCUAAACUUUAA MYC-622: (SEQ ID NO: 3)5′-AGGAACUAUGACCUCGACUACGAct-3′ (SEQ ID NO: 4)3′-UGUCCUUGAUACUGGAGCUGAUGCUGA-5′ MYC-1711: (SEQ ID NO: 5)5′-AGCUUUUUUGCCCUGCGUGACCAga-3′ (SEQ ID NO: 6)3′-CCUCGAAAAAACGGGACGCACUGGUCU-5′where uppercase letters signify to RNA nucleotide, underlined uppercaseletters signify a 2′-O-methyl-RNA nucleotide, and lowercase letterssignify a DNA nucleotide. Note that while SEQ ID NOs: 2, 4, 6, and 8 arepresented above in complementary 3′-5′ orientation, in the SequenceListing provided with this application, they are presented in 5′-3′orientation as required and as shown in listing of Sequences below inTable 11).

Preparation of DsiRNA Strands: Oligonucleotide Synthesis andPurification

Individual RNA strands were synthesized and HPLC purified according tostandard methods (Integrated DNA Technologies, Coralville, Iowa). Forexample, RNA oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected, and desalted on NAP-5 columns(Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques(Damha and Olgivie, Methods Mol. Biol. 20:81, 1993; Wincott et al.,Nucleic Acids Res. 23: 2677, 1995). The oligomers were purified usingion-exchange high performance liquid chromatography (IE-HPLC) on anAmersham Source 15Q column (1.0 cm×25 cm; Amersham Pharmacia Biotech,Piscataway, N.J.) using a 15 min. step-linear gradient. The gradient wasfrom 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A is 100 mMTris pH 8.5 and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Samples weremonitored at 260 nm, and peaks corresponding to the full-lengtholigonucleotide species were collected, pooled, desalted on NAP-5columns, and lyophilized.

The purity of each oligomer was determined by capillary electrophoresis(CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.).The CE capillaries had a 100 μm inner diameter and contained ssDNA 100RGel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide wasinjected into a capillary, run in an electric field of 444 V/cm anddetected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urearunning buffer was purchased from Beckman-Coulter. Oligoribonucleotideswere obtained that are at least 90% pure as assessed by CE for use inexperiments described below. Compound identity was verified bymatrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)mass spectroscopy on a Voyager DETM Biospectometry Workstation (AppliedBiosystems, Foster City, Calif.) following the manufacturer'srecommended protocol. Relative molecular masses of all oligomers wereobtained, often within 0.2% of expected molecular mass.

Preparation of DsiRNA Duplexes

Single-stranded RNA (ssRNA) oligomers were resuspended, e.g., at 100 μMconcentration in duplex buffer consisting of 100 mM potassium acetate,30 mM HEPES, pH 7.5. Complementary sense and antisense strands weremixed in equal molar amounts to yield a final solution of, e.g., 50 μMduplex. Samples were heated to 100° C. for 5 minutes in RNA buffer (IDT)and allowed to cool to room temperature before use. Double-stranded RNA(dsRNA) oligomers were stored at −20° C. Single-stranded RNA oligomerswere stored lyophilized or in nuclease-free water at −80° C.

Preparation of Particles

DsiRNA-lipid complexes were initially produced by combining (a) 24 mg/mLof anti-HPRT1 DsiRNA dissolved in 12.5 mL of water with (b) 37.5 mL of alipid suspension comprising the components of Table 5 in 60 mM HCl(pH=2.3).

TABLE 5 Composition of Aqueous Lipids Used to Form DsiRNA-LipidComplexes DODMA DMPE-PEG2000 MW (Da) 620.90 2693.30 mol % 90.04 9.96 wt% 67.58 32.42 Mol (mmol) 2.14 0.24 Wt (mg) 1330.71 638.31

The above components of Table 5 were extruded through 100 nm membranesfor 10-12 cycles, and were then assessed for particle size andpolydispersity index (PDI), which were 80.76 nm and 0.058, respectively.After combining DsiRNA and the initial lipid suspension, 100 mL of waterwas added, resulting in a final pH of 2.8 for the 150 mL volume ofDsiRNA-lipid complexes obtained (refer to FIG. 1). DsiRNA-lipidcomplexes were then deposited into a mixing vessel, and 100 mL of anadditional solution of lipids (possessing total lipid content of 37mg/mL) dissolved in 100% ethanol was then added to the aqueousDsiRNA-lipid complex suspension. The composition of this additionalsolution of lipids is shown in Table 6.

TABLE 6 Composition of EtOH-Dissolved Lipids Added to DsiRNA-LipidComplexes DSPC CHOL L-30 of Table 1 DSPE-PEG2k MW (Da) 790.16 386.4613.05 2805.50 mol % 19.38 46.44 30.32 3.87 wt % 24.43 28.63 29.65 17.30Mol (mmol) 1.143 2.739 1.788 0.228 Wt (mg) 903.15 1058.35 1096.13 639.65

Upon completing the mixing of additional lipids with the DsiRNA-lipidcomplexes, 500 mL of water were added to the particles, which produced afurther reduction in the ethanol concentration of the mixed suspension,to 14.3% ethanol (optionally, variable volumes of ethanol may be addedat this stage, e.g., 180 mL of water to reduce the ethanol concentrationto 25%, 1.75 L of water to reduce the ethanol concentration to 8%, 3.75L of water to reduce the ethanol concentration to 4%, etc.). This mixedsuspension was then subjected to tangential flow filtration (TFF),resulting in a concentrated volume of about 150 mL. This suspension wasthen diafiltrated with 600 mL PBS, and was then rinsed twice with 50 mLPBS, resulting in a total volume of 220 mL.

Particle size (93.11 nm), polydispersity index (PDI=0.106) andconcentration were then measured. For concentration, DsiRNA-lipidcomplexes were measured as being 1.3 mg/mL, encapsulation efficiency wasobserved to be 95.44% and total volume was 220 mLs, meaning that anaddition of 52.96 mL PBS was required to bring the final concentrationof the sample to 1 mg/mL. Notably, the final ethanol concentration ofthis particle-containing sample was below 0.15%. The processdemonstrated in Example 1 is referred to generally herein as “process2141” and is distinguished in Example 2 from “process 2072” describedbelow.

Example 2 Process 2072 Compared with Process 2141 of Example 1

A process similar to the above process but distinguished from the aboveprocess only in that the concentration of additional lipids in ethanolwas reduced relative to the above-described process (termed the “2072process”) was initially examined for properties of particles so formed.Regarding the processes of this Example, the proportions and totalamounts of lipids used during formulation of particles in the “2072process”, for which results are described in this Example, and the “2141process”, which is set forth in above Example 1, were the same. The onlydifference between the “2141 process” and the “2072 process” can befound in the concentration of lipids that were used in the additionallipids in ethanol solution component of the processes, which waselevated in the “2141 process” in a manner as described in the belowExamples, relative to the “2072 process”.

Particles produced by both the “2072 process” involving addition ofDsiRNA-lipid complexes into additional lipids in ethanol, as well as bythe “2141 process” involving addition of additional lipids in ethanolinto DsiRNA-lipid complexes were assessed for their physical propertiesand compared. The top two panels of FIG. 2 demonstrate that reversal ofthe order of addition of DsiRNA-lipid complexes and additional lipids inethanol (process 2141 as compared with process 2072) created a dramaticand surprising difference in both the average particle size and sizedistribution of particles obtained by the reverse process. Specifically,when a 100 mg batch (DsiRNA content=100 mg) of particles was made by a“2072 process” in which DsiRNA-lipid complexes were added to additionallipids in ethanol, the resultant particles possessed an average size of152.6 nm, while the heterogeneity of this particle population was high,as reflected in an observed PDI value of 0.265 for this preparation. Incontrast, when a larger, 300 mg batch (DsiRNA content=100 mg) ofparticles was made by a process that involved adding the additionallipids in ethanol into the DsiRNA-lipid complex suspension, (the “2141process”) average particle size was reduced to 98.85 and sizedistribution of the particle population was also found to bedramatically more homogeneous (PDI=0.127).

This result demonstrated that the implemented alteration oforder-of-addition impacted particle size and homogeneity in a dramatic,advantageous and surprising manner. As demonstrated in the belowExamples, lower average particle size and reduced heterogeneity of suchparticle populations were associated with both improved efficacy (ofknockdown and phenotypic impact) and improved tolerability/reducedtoxicity of particle populations when they were administered to asubject.

Example 3 Elevating Concentrations of Additional Lipids in EthanolImproved Particle Properties

The above-described process schematically exemplified in FIG. 1, whichis also referred to herein as “2141”, was not only distinguished fromother tested processes in the order of addition of DsiRNA-lipidcomplexes and additional lipids in ethanol, but also was distinguishedfrom other processes by the concentration of lipids and sterols thatwere solubilized in the additional lipids in ethanol solution.Specifically, in the “2072 process”, cholesterol was added to solvent ata concentration of approximately 10-11 mg/ml in ethanol, whichapproached the solubility limit of cholesterol in ethanol. Furtherlipids were then added to this cholesterol-in-ethanol mixture to createthe “additional lipids in ethanol” component, and there was a limit tothe amount of total lipid that could be present in the “additionallipids in ethanol” solution of approximately 20 mg/ml total lipid. Incontrast, in the “2141 process” of the invention, L-30 of Table 1, DSPCand DSPE-PEG2k were combined in 100 ml of ethanol in the amounts shownin Table 6 above. This ethanol solution was then added to cholesterol asa powder, at a cholesterol concentration of approximately 11 mg/ml, butwith the distinction that the total lipid content of this “additionallipids in ethanol” solution achieved a total lipid content of 37 mg/mlin the absence of aggregation or other deleterious effect. (Indeed,additional batches of this “additional lipids in ethanol” solution werealso successfully prepared that possessed approximately 21 mg/mlcholesterol (a level that dramatically exceeded the solubility ofcholesterol alone in ethanol) and 74 mg/ml total lipid in ethanol.)

The impact of driving total lipid content of the “additional lipids inethanol” solution of the current processes above the approximately 20mg/ml or lower levels used for “2072” and similar processes, toapproximately 34 mg/ml in the case of the “2141 process”/particles, wasboth unexpected and dramatic: particles prepared by the “2141 process”possessed improved size and polydispersity as compared to particlesprepared by the “2072 process”; “2141” particles also demonstratedbetter target-specific knockdown than particles prepared by the “2072process”, exhibited improved efficacy at reducing tumor volume in aHep3B mouse model of liver cancer, and were much better tolerated inmice than corresponding particles prepared by the “2072 process”.

The bottom two panels of FIG. 2 show the improved size andpolydispersity values that were observed for a population of particlesprepared using the “2141 process” (which featured an elevatedconcentration of additional lipids in ethanol during the formulationprocess) as compared to such particles prepared using the “2072 process”(which featured total concentrations of additional lipids in ethanol ator below 20 mg/ml during the formulation process). Specifically, the“2141 process” yielded particles that possessed an average size of 95.07nm with observed PDI of 0.072, as compared to corresponding particlesproduced by the “2072 process”, which exhibited an average size of 98.85nm and observed PDI of 0.127. Thus, particles produced by the “2141process” were observed to be slightly more compact and significantlymore homogeneous than corresponding particles produced by the “2072process”, by this gross assessment of the physical properties of bothparticle populations.

Further evaluation of the physical characteristics of “2141”-producedand “2072”-produced particles revealed even more dramatic differencesbetween the two particle populations. When both particle populationswere subjected to size-exclusion chromatography (“SEC”) and signalintensities were examined, the homogeneity of the “2141”-producedparticles was especially striking. As shown in FIG. 3, SEC fractions 2-5of a “2072”-produced particle population revealed significantheterogeneity of the particle population specifically, the “2072”particle population of the top panel of FIG. 3 revealed a significantminor peak of lesser particle size than the main peak, which appeared tocorrespond to micelle debris; in contrast, the “2141”-produced particlesof the bottom panel showed no such minor peak and sizing of“2141”-produced particles was more tightly clustered. Thus, particlesmade by the “2141 process” possessed a more consistent size and lessmicelle debris, as compared to particles produced by the “2072 process”.This effect was particularly noteworthy because, as stated above, thelipid compositions of “2141”-produced and “2072”-produced particles wereidentical.

Additional examination of “2141”-produced particles as compared to“2072”-produced particles further defined the relative homogeneity ofthe “2141”-produced particle population as compared to the heterogeneityof the “2072”-produced particle population. Percent volume analysis wasperformed using 1.0 ml of 1.0 mg/mL DsiRNA in LNP run on a Sepharose 4Bcolumn, 30 ml., and particles were measured by % volume, with RNAcontent for each fraction assessed by average particle size (Malvern).As shown in FIG. 4, when percent volume analysis was performed upon both“2072”- and “2141”-produced particles, “2072”-produced particles (FIG.4, top panel) were observed to possess a majority fraction (57%) of21-32 nm particles within the particle population, apparentlycorresponding to micelle debris, while only 36% of the “2072”-producedparticle population corresponded to the 79-106 nm particles assessed tobe the DsiRNA-containing particles. In contrast, “2141”-producedparticles assessed by percent volume were remarkably homogeneous greaterthan 98% of the particle population was identified in a 51-78 nm windowthat corresponded to DsiRNA-containing particles. Thus, the “2141process” resulted in near-complete incorporation of DsiRNA payload intoproperly-sized particles, whereas the “2072 process” was observed undersuch stringent analyses to have accumulated a significant amount ofmicelle debris within the particle population.

Example 4 The “2141 Process” Produced Particles that Exhibited EnhancedTarget-Specific Knockdown, Improved Phenotypic Activities, and that wereWell-Tolerated

The efficacy of “2141”-produced particles was assessed in vivo in aseries of experiments. First, “2141”-formulated particles harboring ananti-HPRT1 payload DsiRNA as described above were examined fortarget-specific knockdown efficacy in mouse tumors (in such experiments,mice carrying Hep3B tumors were administered 5 mg/kg of particles(“2141” or others as indicated in FIG. 5), and liver tumor knockdown ofHPRT1 was assessed at 48 hours post-administration, N=7/group). As shownin FIG. 5, particles made by the “2141 process” (which is also shared by“2137” and “2144” particles, with only proportions of component lipidsvarying between such these three groups) produced approximately 80%knockdown of HPRT1 in mouse tumors. This result was significantly betterthan that observed for particles made by the “2072 process”, whichresulted in approximately 70% knockdown of the targeted HPRT1 transcriptin mouse tumors. Thus, the “2141 process”, which is distinguished fromthe “2072 process” only in the elevated concentration of lipids presentwithin the “additional lipids in ethanol” component of the process,produced a population of particles that was more effective attarget-specific knockdown of a targeted transcript (here, HPRT1) invivo. Without wishing to be bound by theory, at least part of thisremarkable improvement was likely attributable to the dramaticallyimproved homogeneity of particles obtained via the “2141 process”.

The in vivo phenotypic efficacy of “2141”-formulated particles ascompared to “2072”-formulated particles was also examined. Within suchexperiments, two MYC-targeting DsiRNAs (“MYC-622” and “MYC-1711”) wereformulated using either the “2141 process” or the “2072 process”.Particles containing these MYC-targeting DsiRNAs were then administeredto mice harboring Hep3B tumors and efficacy was assessed (mice weredosed TIW×2 at concentrations indicated in FIG. 6, and tumor weightswere assessed at 48 hours after administration of the final dose). Inprior experiments, both MYC-622 and MYC-1711 payloads were observed topossess comparable efficacies (data not shown). As shown in FIG. 6, aMYC-1711 DsiRNA payload formulated in particles by the “2141 process”exhibited approximately 4-5-fold greater potency (efficacy scaled tolevel of dose) in reducing Hep3B tumor volume in vivo, as compared to“2072”-formulated particles harboring a MYC-622 DsiRNA. Specifically,“2141”-formulated particles harboring MYC-1711 payload administered at 3mg/kg or 5 mg/kg exhibited respective reductions in tumor size of 73%and 77%, respectively. These levels of reduction were significantlygreater than any observed for particles harboring MYC-targeting payloadproduced by the “2072 process”, and even doses of less than 1 mg/kg of“2141”-formulated particles having anti-MYC payloads exhibitedsignificant reductions in tumor size (approximately 28% reduction for0.3 mg/kg and approximately 47% reduction for 0.5 mg/kg). Tolerabilityof the respective formulations was also assessed by examining thefollowing toxicity markers: ALK Phos, ALT, AST, ALB and total bilirubin.As shown in the lower table of FIG. 6, particles produced by the “2072process” raised levels of such toxicity markers to a much greater extentthan was observed for particles produced by the “2141 process”.

Additional assays were performed to assess the tolerability of particlesproduced by either the “2141 process” or the “2072 process”, and allsuch assays underscored the enhanced tolerability/lack of toxicity ofparticles produced by the “2141 process”. When a formulation is not welltolerated in a mouse, such a mouse will often show a loss of body weightfollowing administration of such a formulation. Meanwhile, liver weightoften increases for such mice. As shown in FIG. 7, gross tolerabilitiesof “2072”- and “2141”-produced particles were compared via evaluation ofthe impact upon body weight and liver weight of 10 mg/kg administration(BIW×2, four doses total, n=15/group) of such particles to mice.Remarkable differences between particles formulated by each of theseprocesses were observed, with “2072”-formulated particles exhibiting adramatic impact upon both body weight (administration of“2072”-formulated particles produced a reduction in body weight ofalmost 20%) and liver weight (an approximate 50% increase in liverweight was observed for mice administered the “2072”-formulatedparticles. In contrast, no significant impact upon body weight wasobserved for mice administered “2141”-formulated particles, and“2141”-formulated particles provoked only a very modest (approx. 10-20%)increase in liver weight, an effect that was also only observed in oneof two “2141” preparations examined. Thus, “2141”-produced particleswere remarkably well-tolerated, based upon gross indications offormulation tolerability, in vivo.

Such tolerability/toxicity results of the “2141”-formulated particleswere reinforced by assessment of the following markers of toxicity inthose mice administered the “2072”- and “2141”-formulated particles at10 mg/kg administration (BIW×2, four doses total, n=15/group): ALT, AST,bilirubin, CPK, alkaline phosphatase, and albumin. As shown in FIG. 8,such elevated, repeated doses of “2072”-formulated particles provokedchanges in each of these markers that were indicative of formulationtoxicity: ALT, AST, bilirubin, CPK and alkaline phosphatase levels wereall significantly elevated, while albumin levels were significantlyreduced. In stark contrast, “2141”-formulated particles showed no suchdramatic changes in the toxicity markers examined: none of the“2141”-formulated particles showed any significant change in suchtoxicity marker, as compared to parallel PBS-treated animals. Thus, the“2141 process” (which featured an elevated concentration of additionallipids in ethanol during the formulation process) produced particlesthat not only possessed improved physical characteristics (size, PDI,etc.), but also were more efficacious in vivo, as well as being bettertolerated, than corresponding particles produced by methods that did notfeature the use of elevated concentrations of additional lipids inethanol during the formulation process.

Example 5 Processes and Formulations for Producing AnionicAgent-Containing Particles

Lipid compositions for use in the production of particles carryinganionic agents were formulated generally as described above for Examples1 (the 2141 process) and according to the process shown in FIG. 1. Theprocess includes preparing a first lipid suspension comprising corelipids including a cationic lipid such as DODMA, DL-048, DL-049, DL-033,and a modified lipid which prevents particle aggregation duringlipid-anionic agent particle formulation, for example, a PEG-lipidconjugate such as DMPE-PEG2k, DMG-PEG2k, and DSPE-PEG2k. The core lipidsare mixed in an acidic aqueous solution to form a lipid complex.

A second (additional) lipid solution is prepared in a solvent, forexample, ethanol, preferably 100% ethanol. As shown Tables 7 and 8, thesecond lipid solution contains one or more lipid selected from the groupconsisting of a structural lipid, a sterol, a cationic lipid, and amodified lipid. Examples include DDPC, DSPC, MSPC, POPC, Lyso PC, POGP,Cholesterol, DL-033, DL-036, DMPD-PEG2k, DSPE-PEG2k, and DSG-PEG2k, andthe like. Chemical abbreviations of compounds used in the formulationsare defined below in Table 9.

Specific exemplary combinations of lipids for preparing the core firstlipid composition and the second additional lipid compositions arelisted in Tables 7 and 8, and were prepared as described above forExample 1 and tested for specific properties. As described for Example1, a preferred method for producing particles containing an anionicagent payload includes the steps of combining in an acidic aqueoussolution, preferably an aqueous HCl solution, a modified lipid whichprevents particle aggregation during lipid-anionic agent particleformation and a cationic lipid (for example, the core cationic lipidsshown in Tables 7 and 8), in amount sufficient to form a complex. Thelipid complex can then be combined with an anionic agent, such as anucleic acid molecule, to form a complex-anionic agent, and combinedwith a neutral aqueous solution to form a complex-anionic agent aqueoussuspension.

Additional lipids are combined to form an additional lipid solution orsuspension, comprising one or more lipid selected from a structurallipid, a sterol, a cationic lipid, and a modified lipid. In a preferredembodiment, the additional lipids are combined in a solvent such asethanol, preferably 100% ethanol, and preferably include one or more ofthe Envelope lipids shown in Tables 7 and 8. The solution or suspensionof the additional Envelope lipids is preferably added to thecomplex-anionic agent to produce particles comprising the anionic agent.

Particles containing an antigentic agent were produced using theformulations described in Tables 7 and 8 and each of the formulationsprovided particles having the improved characteristics described forparticles produced by the 2141 process as compared against particlesmade by the 2072 process described in Examples 1 and 2. The improvedcharacteristics included homogenicity, uniformity, payload, andtherapeutic efficacy.

As demonstrated in Table 10, the particles have improved characteristicsover particles produced by the process of 2072 as measured by one ormore of the following characteristics and/or markers: Average particlesize, polydispersity index (PDI), percent payload, for example, nucleicacid in the particle and/or delivered to the target cell, diseasemarkers alkaline phosphatase, CPK, ALT, AST, Albumin, total bilirubin,HPRT1, change in body weight or liver weight, for example, as measuredin diagnostic assays representative of the payload and its target. SeeTable 10, below.

TABLE 7 EnCore LNP Formulation Compositions (% mol) - Tumor CentricTumor Centric EnCore LNP Formulation Compositions (% mol) Lipid 21412163 2311 2332 2376 Core DODMA 25.9 Lipids DL-048 25.9 25.9 DL-049DL-033 25.2 24.7 DMPE-PEG2k 2.9 2.9 DMG-PEG2k DSPE-PEG2k 2.8 2.7 2.9Total 28.7 28.0 28.7 27.4 28.7 Envelope DPPC Lipids DSPC 13.8 13.4 13.813.2 13.8 MSPC POPC Lyso PC POPG CHOL 33.1 32.2 33.1 31.6 33.1 DL-03321.0 DL-036 21.6 21.6 20.6 21.6 DMPE-PEG2k DSPE-PEG2k 2.8 2.8 2.8DSG-PEG2k 5.3 7.3 Total 71.3 72.0 71.3 72.6 71.3 Grand Total 100.00100.0 100.0 100.00 100.00 PC lipids (zwitterionic) 13.8 13.4 13.8 13.213.8 PG lipids 0.0 0.0 0.0 0.0 0.0 PO4-Group 19.4 16.2 19.4 15.9 19.4Asymmetric Lipids 0.0 0.0 0.0 0.0 0.0 Ionizable (Positive) 47.5 46.247.5 45.3 47.5 Ionizable (Negative) N/A N/A N/A N/A N/A PEG Lipids 5.68.1 5.6 10.0 5.6

TABLE 8 Target/Encore Liver Centric # 2185/2325 2345 2357 2360 2361 23622363 2368 2372 2373 Core DODMA 24.7 27.6 31.2 35.8 DL-048 26.6 25.3 25.316.3 27.8 DL-049 25.3 DL-033 DMPE- 2.9 2.8 2.8 2.7 3.0 3.4 4.0 PEG2kDMG- 2.8 1.8 3.1 PEG2k DSPE- PEG2k Total 29.6 28.1 28.1 27.4 30.6 34.639.8 28.1 18.1 30.9 ENV DPPC 14.2 DSPC MSPC POPC Lyso PC POPG CHOL 34.036.0 36.0 36.3 34.7 32.7 30.1 36.0 41.0 34.6 DL-033 DL-036 22.2 36.036.0 36.3 34.7 32.7 30.1 36.0 41.0 34.6 DMPE- PEG2k DSPE- PEG2k DSG-PEG2k Total 70.4 71.9 71.9 72.6 69.4 65.4 60.2 71.9 81.9 69.1 GrandTotal 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0Target/Encore Liver Centric # 2386 2391 2408 2410 2411 2413 2414 2416Core DODMA DL-048 24.6 24.3 26.8 29.9 26.8 29.9 26.8 29.9 DL-049 DL-033DMPE- 2.7 2.5 3.0 3.3 3.0 3.3 3.0 3.3 PEG2k DMG- PEG2k DSPE- PEG2k Total27.3 26.8 29.7 33.2 29.7 33.2 29.7 33.2 ENV DPPC DSPC MSPC 14.1 3.9 POPC14.1 3.9 Lyso PC 14.1 3.9 POPG 0.4 2.2 CHOL 36.2 35.5 33.7 37.7 33.737.7 33.7 37.7 DL-033 DL-036 36.2 35.5 22.5 25.1 22.5 25.1 22.5 25.1DMPE- PEG2k DSPE- PEG2k DSG- PEG2k Total 72.7 73.2 70.3 66.8 70.3 66.870.3 66.8 Grand Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

TABLE 9 Chemical Names Abbreviation Chemical name CAS # DPPC1,2-dipalmitoyl-sn-glycero-3-phosphocholine 63-89-8 DSPC1,2-distearoyl-sn-glycero-3-phosphocholine 816-94-4 MSPC1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine 76343-22-1 POPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 26853-31-6 Lyso PC1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine 17364-16-8 POPG1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1′-rac-268550-95-4 glycerol) CHOL cholesterol 57-88-5 DMPE-PEG2k1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- 474922-82-2[methoxy(polyethylene glycol)-2000] DMG-PEG2k1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol-2000 DSPE-PEG2k1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- 474922-77-5[methoxy(polyethylene glycol)-2000] DSG-PEG2k1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol-2000 308805-39-2DODMA 1,2-dioleyloxy-N,N-dimethylaminopropane 104162-47-2 DL-048Dioleyl-N,N-DimethylGlycine DL-049 Dioleyl-N,N-DimethylGlycine DL-033DiLin-N-Methylpiperazine DL-036 DiLin-N,N-DimethylGlycine

TABLE 10 Tumor Liver % RNA KD₅₀ KD₅₀ Formulation PSD (nm) PDI EncapComposition (mpk) (μg/kg) Note 2141 100 ± 10  ≦0.15 ≧85% DODMA DMPE-DSPC CHOL DL-036 DSPE- 0.5 to 1 PEG2k PEG2k in HCC 2163 90 ± 10 ≦0.15≧85% DL-033 DSPE- DSPC CHOL DL-033 DSG- <0.5 in PEG2k PEG2k HCC 2311 100± 10  ≦0.15 ≧85% DL-048 DMPE- DSPC CHOL DL-036 DSPE- 0.5 to 1 PEG2kPEG2k in HCC 2332 90 ± 10 ≦0.15 ≧85% DL-033 DSPE- DSPC CHOL DL-033 DSG-to 5 Very high PEG2k PEG2k in Prostate PEG content 2376 100 ± 10  ≦0.15≧85% DL-048 DSPE- DSPC CHOL DL-036 DSPE- 0.5 to 1 PEG2k PEG2k in HCC2185/2325 100 ± 10  ≦0.15 ≧85% DL-048 DMPE- DPPC CHOL DL-036 N/A N/A 20to 50 PEG2k 2345 105 ± 15  ≦0.15 ≧85% DL-048 DMPE- N/A CHOL DL-036 N/AN/A 10 to 20 PC free PEG2k 2357 105 ± 15  ≦0.15 ≧85% DL-049 DMPE- N/ACHOL DL-036 N/A N/A 10 to 20 PC free PEG2k 2360 105 ± 15  ≦0.15 ≧85%DODMA DMPE- N/A CHOL DL-036 N/A N/A 10 to 20 PC free PEG2k 2368 105 ±15  ≦0.15 ≧85% DL-048 DMG- N/A CHOL DL-036 N/A N/A 10 to 20 PO4- groupfree PEG2k 2386 105 ± 15  ≦0.15 ≧85% DL-048 DMG- POPG (0.5%) CHOL DL-036N/A N/A 10 to 20 PG containing PEG2k 2391 105 ± 15  ≦0.15 ≧85% DL-048DMG- POPG (3%) CHOL DL-036 N/A N/A 10 to 20 PG containing PEG2k 2408 100± 15  ≦0.15 ≧85% DL-048 DMPE- POPG (3%) CHOL DL-036 N/A N/A 20 to 50Asymmetric PEG2k acyl chains 2410 100 ± 15  ≦0.15 ≧85% DL-048 DMPE- POPC(5%) CHOL DL-036 N/A N/A 20 to 50 Same as above PEG2k 2411 100 ± 15 ≦0.15 ≧85% DL-048 DMPE- MSPC (20%) CHOL DL-036 N/A N/A 20 to 50 Same asabove PEG2k 2413 100 ± 15  ≦0.15 ≧85% DL-048 DMPE- MSPC (5%) CHOL DL-036N/A N/A 20 to 50 Same as above PEG2k 2414 100 ± 15  ≦0.15 ≧85% DL-048DMPE- Lyso PC CHOL DL-036 N/A N/A 20 to 50 Same as above PEG2k (C16,20%) 2416 100 ± 15  ≦0.15 ≧85% DL-048 DMPE- Lyso PC CHOL DL-036 N/A N/A20 to 50 Same as above PEG2k (C16, 5%)

Example 6 Liver Centric Formulations: HAO1 Knockdown

Additional lipid-nucleic acid formulations were prepared as describedfor example 1 according to the 2041 process and tested for effectiveproduction of therapeutic particles, and to determine a minimum dose andfrequency sufficient for HAO1 gene and protein knockdown. DsiRNAtargeting HAO was formulated in EnCore lipid particles according to thespecific formulations shown below in Table 11 and prepared as describedfor Example 1, the 2141 process. Formulations 2401 and 2373 containedthe same combination of lipids and nucleic acid agent, but differed inthat the 2401 process included in-line mixing of the envelope lipidsolution or suspension with the complex-anionic agent aqueoussuspension, whereas the 2373 process utilized batch mixing.

Particles were injected intravenously into female mice and animals weresacrificed 24 and 168 hours post dosing. Plasma and liver tissue sampleswere collected for analysis of hydroxyacid oxidase 1 (HAO1) gene andprotein expression. The targeting DsiRNA was an RNAi agent (DsiRNA forMAO1), having the following sequences, respectively SEQ ID NO: 7 and 8.Note that while Antisense SEQ ID NO: 8 is shown below in 3′-5′orientation, in the Sequence Listing, all sequences are presented in5′-3′ orientation.

Sense: (SEQ ID NO: 7) 5′-rAmUrAmUrUmUrUrCrCrCrArUrCmUrGmUrAmUrUrArUrUrUTT-3′ AntiSense: (SEQ ID NO: 8)*3′-mAmAmAmArArUrArAmUrAmCrAmGrAmUrGrGrGrArArArAm UrAmUmUmG-5′

In this study, the efficacy of particles produced with POPG as anenvelope lipid was compared with DPPC. Particles contained variedamounts of POPG from 0.5% to 3%, while other components, includingenvelope lipids Cholesterol and DL-036 and Core lipids DL-048 andDMG-PEG2k were constant across all formulations. See Table 10 below.

Particles were prepared as disclosed above for Examples 1 and 5, usingthe specific formulations described in Table 11 below, and DsiRNA formedfrom SEQ ID NOs: 7 and 8, which was designed to interfere with thecellular target, MAO1.

For this study, formulations containing murine FVII DsiRNA payload wereintravenously injected into CB57-BL6 female mice at 10 ug/kg (circles),25 ug/kg (diamonds), or 50 ug/kg (squares) as indicated in FIG. 9. Serumsamples were collected 24 hour post dosing to access the reduction FVIIprotein using activity assay. PBS was included as negative control.Different EnCore formulations were labeled with individual 4 digitnumbers as indicated in the Table 11 and FIG. 9. Different compositionsof lipids were used for each formulation as shown on the bottom of theFigure.

The particles were tested for improved efficacy by analyzing presence ofFactor VII in the mice receiving the interfering therapeutic moleculevia the lipid particles. The assay measured conversion of human factorX, a substrate of Factor VII, to Factor Xa, a reaction that then actedupon sXa-11, a chromogenic substrate, to produce color measured at 405nm. The data are shown in Table 11 and FIG. 9.

TABLE 11 Formulations for Liver Centric Efficacy Screen Liver CentricTarget/ 2185/ Encore # 2325 2345 2368 2373 2386 3287 2388 2389 2390 23912401 Core DL-048 26.6 25.3 25.3 27.8 27.0 26.9 26.8 26.7 26.6 26.5 27.8DMPE-PEG2k 2.9 2.8 DMG-PEG2k 2.8 3.1 3.0 3.0 3.0 3.0 3.0 3.0 3.1 Total29.6 28.1 28.1 30.9 30.0 29.9 29.8 29.7 29.6 29.5 30.9 ENV DPPC 14.2POPG 0.4 0.8 1.1 1.4 1.8 2.1 CHOL 34.0 36.0 36.0 34.6 34.8 34.7 34.534.4 34.3 34.2 34.6 DL-036 22.2 36.0 36.0 34.6 34.8 34.7 34.5 34.4 34.334.2 34.6 Total 70.4 71.9 71.9 69.1 70.0 70.1 70.2 70.3 70.4 70.45 69.1Grand Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0100.0

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

Listing of the Sequences disclosed herein:

SEQ ID NA/ NO: Protein Species Sequence 5′-3′ * 1 NA syntheticGCCAGACUUUGUUGGAUUUGAAAtt 2 NA synthetic AAUUUCAAAUCCAACAAAGUCUGGCUU 3NA synthetic AGGAACUAUGACCUCGACUACGAct 4 NA syntheticUGUCCUUGAUACUGGAGCUGAUGCUGA 5 NA synthetic AGCUUUUUUGCCCUGCGUGACCAga 6NA synthetic CCUCGAAAAAACGGGACGCACUGGUCU 7 NA syntheticrAmUrAmUrUmUrUrCrCrCrArUrCmU rGmUrAmUrUrArUrUrUTT 8 NA syntheticGUmUmAmUrAmArArArGrGrGrUrAmG rAmCrAmUrAmArUrArArmAmAmAm * whereUPPERCASE letters signify to RNA nucleotide, underlined uppercaseletters signify a 2′-O-methyl-RNA nucleotide, and lowercase letterssignify a DNA nucleotide.

We claim:
 1. A method of producing a particle comprising an anionicagent comprising: (a) combining in an acidic aqueous solution (i) amodified lipid which prevents particle aggregation during lipid-anionicagent particle formation and (ii) a cationic lipid, in an amountsufficient for a complex to form; (b) combining the complex of step (a)with an anionic agent; (c) combining a neutral aqueous solution with thecomplex-anionic agent of step (b) to form a complex-anionic agentaqueous suspension; (d) forming a solution or suspension comprising atleast one lipid selected from the group consisting of a structurallipid, a sterol, a cationic lipid and a modified lipid which preventsparticle aggregation during lipid-anionic agent particle formation; and(e) combining the solution or suspension of step (d) with thecomplex-anionic agent aqueous solution of step (c) by a method selectedfrom the group consisting of adding the solution or suspension of step(d) to the complex-anionic agent aqueous suspension of step (c) andin-line mixing the solution or suspension of step (d) and thecomplex-anionic agent aqueous solution of step (c), or (f) adding thesolution or suspension of step (d) to the complex-anionic agent aqueoussuspension of step (c) thereby producing a particle comprising ananionic agent.
 2. The method of claim 1, wherein said acidic aqueoussolution of step (a) comprises HCl.
 3. The method of claim 1, whereinsaid acidic aqueous solution of step (a) possesses a pH of less than 4.4. The method of claim 1, wherein said acidic aqueous solution of step(a) is about 60 mM HCl.
 5. The method of claim 1, wherein said cationiclipid of step (a) comprises a protonatable group.
 6. The method of claim5, wherein said cationic lipid of step (a) has a pKa of from 4 to
 11. 7.The method of claim 1, wherein said cationic lipid of step (a) isselected from the group consisting of DODMA, DOTMA and a cationic lipidof Table
 1. 8. The method of claim 1, wherein said modified lipid whichprevents particle aggregation during lipid-anionic agent particleformation of step (a) or step (d) is a PEG-lipid.
 9. The method of claim1, wherein said modified lipid of step (a) or step (d) is selected fromthe group consisting of DMPE-PEG, DSPE-PEG and DSG-PEG.
 10. The methodof claim 8, wherein said PEG is PEG2k.
 11. The method of claim 1,wherein the complex of step (a) is between 60 and 75 nM in diameter. 12.The method of claim 1, wherein said anionic agent of step (b) is apolyanionic agent
 13. The method of claim 1, wherein said anionic agentis a nucleic acid.
 14. The method of claim 13, wherein said nucleic acidis selected from the group consisting of an antisense oligonucleotideand a double-stranded nucleic acid.
 15. The method of claim 14, whereinsaid double-stranded nucleic acid is selected from the group consistingof a small hairpin RNA (shRNA) and a siRNA.
 16. The method of claim 15,wherein said double-stranded nucleic acid is a substrate for humanDicer.
 17. The method of claim 1, wherein said neutral aqueous solutionof step (c) is water.
 18. The method of claim 1, wherein said forming asolution or suspension of step (d) involves dissolving in ethanol saidat least one lipid selected from the group consisting of a structurallipid, a sterol, a cationic lipid and a modified lipid which preventsparticle aggregation during lipid-anionic agent particle formation. 19.The method of step 18, wherein said forming a solution or suspension ofstep (d) involves dissolving in 100% ethanol.
 20. The method of claim 1,wherein said structural lipid of step (d) is selected from the groupconsisting of DSPC, DPPC and DOPC.
 21. The method of claim 1, whereinsaid sterol is cholesterol.
 22. The method of claim 1, wherein saidcationic lipid of step (d) is selected from Table
 1. 23. The method ofclaim 1, wherein said particle comprising an anionic agent is between 90and 110 nm in diameter.
 24. The method of claim 1, wherein said particlecomprising an anionic agent is made at a scale selected from the groupconsisting of 10 mg or more of anionic agent, 50 mg or more of anionicagent, 100 mg or more of anionic agent, 250 mg or more of anionic agent,500 mg or more of anionic agent, 1 g or more of anionic agent, 2 g ormore of anionic agent, 3 g or more of anionic agent, 4 g or more ofanionic agent, 5 g or more of anionic agent, 7.5 g or more of anionicagent, 10 g or more of anionic agent, 20 g or more of anionic agent, 40g or more of anionic agent, 50 g or more of anionic agent, 100 g or moreof anionic agent, 200 g or more or anionic agent, 300 g or more ofanionic agent, 400 g or more of anionic agent, 500 g or more of anionicagent, 1 kg or more of anionic agent, 2 kg or more of anionic agent, 3kg or more of anionic agent, 4 kg or more of anionic agent, 5 kg or moreof anionic agent and 10 kg or more of anionic agent.
 25. The method ofclaim 1, wherein said particle comprising an anionic agent possesses aproperty selected from the group consisting of improved size and/or PDI,improved efficacy in a subject administered said particle and improvedtolerability in a subject administered said particle, as compared to anappropriate control particle formed by an appropriate control processthat comprises combining the solution or suspension of step (d) with thecomplex-anionic agent aqueous solution of step (c) by adding thecomplex-anionic agent aqueous suspension of step (c) to the solution orsuspension of step (d).
 26. The method of claim 1, further comprisingthe following step: (f) combining the particle comprising an anionicagent with a volume of water sufficient to reduce the concentration ofethanol within the combined solution to 10% or less.
 27. The method ofclaim 26, further comprising the step of: (g) performing a processselected from the group consisting of tangential flow filtration (TFF)and dialysis upon said combined solution.
 28. The method of claim 27,wherein said combined solution is dialyzed against PBS.
 29. A method ofproducing a particle comprising an anionic agent comprising: (a)combining in an acidic aqueous solution (i) a modified lipid whichprevents particle aggregation during lipid-anionic agent particleformation and (ii) a cationic lipid, in an amount sufficient for acomplex to form; (b) combining the complex of step (a) with an anionicagent; (c) combining a neutral aqueous solution with the complex-anionicagent of step (b) to form a complex-anionic agent aqueous suspension;(d) forming a solution or suspension comprising at least one lipidselected from the group consisting of a structural lipid, a sterol, acationic lipid and a modified lipid which prevents particle aggregationduring lipid-anionic agent particle formation; and (e) adding thesolution or suspension of step (d) to the complex-anionic agent aqueoussuspension of step (c), thereby producing a particle comprising ananionic agent.
 30. A method for increasing the solubility of a firstlipid or sterol in a solvent comprising: combining a second lipid orsterol with said solvent to form a second lipid or sterol solution insaid solvent, wherein said solvent is free of said first lipid; andcombining said second lipid or sterol solution in said solvent with saidfirst lipid or sterol to form a solution of the second lipid or steroland the first lipid or sterol, wherein the solubility of said firstlipid or sterol in said solvent in the presence of said second lipid orsterol is higher than the solubility of said first lipid or sterol insaid solvent in the absence of said second lipid or sterol, therebyincreasing the solubility of the first lipid or sterol in said solvent.31. A method for increasing the solubility of a first lipid or sterol ina solvent comprising: combining a second lipid or sterol with said firstlipid or sterol in the absence of a solvent, wherein the solubility ofsaid first lipid or sterol in said solvent in the presence of saidsecond lipid or sterol is higher than the solubility of said first lipidor sterol in said solvent in the absence of said second lipid or sterol,thereby increasing the solubility of the first lipid or sterol in saidsolvent.
 32. The method of claim 30, wherein said first lipid or sterolis a sterol selected from the group consisting of cholesterol,cholestanone, cholestenone, coprostanol,3β-[-(N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-cholesterol) and bis-guanidium-tren-cholesterol (BGTC).
 33. Themethod of claim 30, wherein said first lipid or sterol is present at aconcentration selected from the group consisting of 10 mg/ml or more, 11mg/ml or more, 12 mg/ml or more, 15 mg/ml or more, 20 mg/ml or more, 25mg/ml or more, 30 mg/ml or more, 35 mg/ml or more, 37 mg/ml or more, 40mg/ml or more, 45 mg/ml or more, 50 mg/ml or more, 55 mg/ml or more, 60mg/ml or more, 65 mg/ml or more, 70 mg/ml or more, 74 mg/ml or more, 75mg/ml or more, 80 mg/ml or more, 85 mg/ml or more, 90 mg/ml or more, 95mg/ml or more, 100 mg/ml or more, 150 mg/ml or more, 200 mg/ml or more,250 mg/ml or more, 500 mg/ml or more and 1 g/ml or more within saidsolution of the second lipid or sterol and the first lipid or sterol.34. The method of claim 30, wherein the total lipid content of saidsolution of the second lipid or sterol and the first lipid or sterol isselected from the group consisting of 12 mg/ml or more, 15 mg/ml ormore, 20 mg/ml or more, 25 mg/ml or more, 30 mg/ml or more, 35 mg/ml ormore, 37 mg/ml or more, 40 mg/ml or more, 45 mg/ml or more, 50 mg/ml ormore, 55 mg/ml or more, 60 mg/ml or more, 65 mg/ml or more, 70 mg/ml ormore, 74 mg/ml or more, 75 mg/ml or more, 80 mg/ml or more, 85 mg/ml ormore, 90 mg/ml or more, 95 mg/ml or more, 100 mg/ml or more, 150 mg/mlor more, 200 mg/ml or more, 250 mg/ml or more, 500 mg/ml or more and 1g/ml or more.
 35. The method of claim 30, wherein said first lipid orsterol is selected from Table
 1. 36. The method of claim 30, whereinsaid second lipid or sterol is selected from Table
 1. 37. The method ofclaim 30, wherein said second lipid or sterol solution in said solventcomprises one or more additional lipids selected from Table
 1. 38. Themethod of claim 30, wherein said solvent is selected from the groupconsisting of ethanol, saline, methanol, n-propanol, isopropanol,n-butanol, isobutanol, tert-butanol, glycerol, ethylene glycol,propylene glycol, polyethylene glycol, chloroform, dichloromethane,hexane, cyclohexane, acetone, ether, diethyl ether, dioxan, isopropylether, tetrahydrofuran and combinations thereof.
 39. The method of claim30, wherein said solvent is selected from the group consisting of aceticacid, acetone, acetonitrile, anisole, benzene, 1-butanol, 2-butanol,butyl acetate, tert-butylmethyl ether, carbon tetrachloride,chlorobenzene, chloroform, cumene, cyclohexane, 1,2-dichloroethane,1,1-dichloroethene, 1,2-dichloroethene, dichloromethane,1,2-dimethoxyethane, n,n-dimethylacetamide, n,n-dimethylformamide,dimethyl sulfoxide, 1,4-dioxane, ethanol, 2-ethoxyethanol, ethylacetate, ethyleneglycol, ethyl ether, ethyl formate, formamide, formicacid, heptane, hexane, isobutyl acetate, isopropyl acetate, methanol,2-methoxyethanol, methyl acetate, 3-methyl-1-butanol, methylbutylketone, methylcyclohexane, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, n-methylpyrrolidone, nitromethane, pentane,1-pentanol, 1-propanol, 2-propanol, propyl acetate, pyridine, sulfolane,tetrahydrofuran, tetralin, toluene, 1,1,1-trichloroethane,1,1,2-trichloroethene, xylene, and combinations thereof.
 40. The methodof claim 30, wherein said solvent is ethanol.
 41. The method of claim30, wherein said solution of the second lipid or sterol and the firstlipid or sterol comprises at least one lipid selected from the groupconsisting of a structural lipid, a sterol, a cationic lipid and amodified lipid which prevents particle aggregation during lipid-anionicagent particle formation.
 42. A method of producing a particlecomprising a first lipid or sterol, a second lipid or sterol and a smallmolecule comprising: (a) combining a second lipid or sterol with asolvent to form a second lipid or sterol solution in said solvent,wherein said solvent is free of said first lipid or sterol; and (b)combining said second lipid or sterol solution in said solvent with saidfirst lipid or sterol to form a solution of the second lipid or steroland the first lipid or sterol, wherein the solubility of said firstlipid or sterol in said solvent in the presence of said second lipid orsterol is higher than the solubility of said first lipid or sterol insaid solvent in the absence of said second lipid or sterol, (c)combining the solution of the second lipid or sterol and the first lipidor sterol of step (b) with a small molecule, thereby producing aparticle comprising a first lipid or sterol, a second lipid or steroland a small molecule.
 43. A method of producing a particle comprising afirst lipid or sterol, a second lipid or sterol and an anionic agentcomprising: (a) combining a second lipid or sterol with a solvent toform a second lipid or sterol solution in said solvent, wherein saidsolvent is free of said first lipid or sterol; and (b) combining saidsecond lipid or sterol solution in said solvent with said first lipid orsterol to form a solution of the second lipid or sterol and the firstlipid or sterol, wherein the solubility of said first lipid or sterol insaid solvent in the presence of said second lipid or sterol is higherthan the solubility of said first lipid or sterol in said solvent in theabsence of said second lipid or sterol, (c) combining the solution ofthe second lipid or sterol and the first lipid or sterol of step (b)with an anionic agent, thereby producing a particle comprising a firstlipid or sterol, a second lipid or sterol and an anionic agent.
 44. Amethod of producing a particle comprising a first lipid or sterol, asecond lipid or sterol and an anionic agent comprising: (a) combining inan acidic aqueous solution (i) a modified lipid which prevents particleaggregation during lipid-anionic agent particle formation and (ii) acationic lipid, in an amount sufficient for a complex to form; (b)combining the complex of step (a) with an anionic agent; (c) combining aneutral aqueous solution with the complex-anionic agent of step (b) toform a complex-anionic agent aqueous suspension; (d) combining a secondlipid or sterol with a solvent to form a second lipid or sterol solutionin said solvent, wherein said solvent is free of said first lipid orsterol; and (e) combining said second lipid or sterol solution in saidsolvent with said first lipid or sterol to form a solution of the secondlipid or sterol and the first lipid or sterol, (f) combining thesolution of the second lipid or sterol and the first lipid or sterol ofstep (e) (d) with the complex-anionic agent aqueous solution of step(c), thereby producing a particle comprising a first lipid or sterol, asecond lipid or sterol and an anionic agent.
 45. The method of claim 44,wherein the solubility of said first lipid or sterol in said solvent inthe presence of said second lipid or sterol in step (e) is higher thanthe solubility of said first lipid or sterol in said solvent in theabsence of said second lipid or sterol.
 46. The method of claim 43,wherein said solution of the second lipid or sterol and the first lipidor sterol comprises at least one lipid selected from the groupconsisting of a structural lipid, a sterol, a cationic lipid and amodified lipid which prevents particle aggregation during lipid-anionicagent particle formation.
 47. The method of claim 43, wherein saidparticle possesses a property selected from the group consisting ofimproved size and/or PDI, improved efficacy in a subject administeredsaid particle and improved tolerability in a subject administered saidparticle, as compared to an appropriate control particle formed by anappropriate control process that comprises exposing said first lipid orsterol to said solvent before said second lipid or sterol is exposed tosaid solvent.
 48. The method of claim 43, wherein said anionic agent isa nucleic acid.
 49. The method of claim 48, wherein said nucleic acid isselected from the group consisting of an antisense oligonucleotide and adouble-stranded nucleic acid.
 50. The method of claim 49, wherein saiddouble-stranded nucleic acid is selected from the group consisting of asmall hairpin RNA (shRNA) and a siRNA.
 51. The method of claim 50,wherein said double-stranded nucleic acid is a substrate for humanDicer.
 52. The method of claim 44, wherein step (f) is performed by amethod selected from the group consisting of adding the solution of thesecond lipid or sterol and the first lipid or sterol of step (d) to thecomplex-anionic agent aqueous suspension of step (c) and in-line mixingthe solution of the second lipid or sterol and the first lipid or sterolof step (d) and the complex-anionic agent aqueous solution of step (c).